Exogenously triggered controlled release materials and uses thereof

ABSTRACT

The disclosure provides cross-linked materials that include multivalent cross-linking agents that bind an exogenous target molecule; and conjugates that include two or more separate affinity ligands bound to a conjugate framework, wherein the two or more affinity ligands compete with the exogenous target molecule for binding with the cross-linking agents and wherein conjugates are cross-linked within the material as a result of non-covalent interactions between cross-linking agents and affinity ligands on different conjugates. The conjugates also include a drug.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/147,878 filed Jan. 28, 2009, U.S. Provisional Application No.61/159,643 filed Mar. 12, 2009, U.S. Provisional Application No.61/162,107 filed Mar. 20, 2009, U.S. Provisional Application No.61/162,053 filed Mar. 20, 2009, U.S. Provisional Application No.61/162,058 filed Mar. 20, 2009, U.S. Provisional Application No.61/162,084 filed Mar. 20, 2009, U.S. Provisional Application No.61/162,092 filed Mar. 20, 2009, U.S. Provisional Application No.61/162,105 filed Mar. 20, 2009, U.S. Provisional Application No.61/163,084 filed Mar. 25, 2009, U.S. Provisional Application No.61/219,897 filed Jun. 24, 2009, U.S. Provisional Application No.61/223,572 filed Jul. 7, 2009, and U.S. Provisional Application No.61/252,857 filed Oct. 19, 2009, the content of each of which is herebyincorporated by reference in its entirety.

BACKGROUND

The majority of “controlled-release” drug delivery systems operate byslowing or delaying the release of a drug post-administration. Whilethese systems are useful for certain types of drugs (e.g., because theylead to fewer peaks and troughs in the serum profile, reducedside-effects, etc.) they are unsuitable for drugs that require morecomplex release profiles (e.g., release in proportion to an endogenoussubstance such as glucose, pulsatile release at fixed or variable timepoints, etc). For example, the treatment of diabetes mellitus withinjectable insulin is a well-known and studied case where gradual slowrelease of insulin is ineffective. In fact, it is apparent that thesimple replacement of the hormone is not sufficient to prevent thepathological sequelae associated with this disease. The development ofthese sequelae is believed to reflect an inability to provide exogenousinsulin proportional to varying blood glucose concentrations experiencedby the patient (i.e., a truly “controlled-release” system). As a result,there remains a need in the art for alternative controlled-release drugdelivery systems and in particular systems that can be controlledpost-administration. The present disclosure provides such systems.

SUMMARY

In one aspect, the disclosure provides cross-linked materials thatinclude multivalent cross-linking agents that bind an exogenous targetmolecule; and conjugates that include two or more separate affinityligands bound to a conjugate framework, wherein the two or more affinityligands compete with the exogenous target molecule for binding with thecross-linking agents and wherein conjugates are cross-linked within thematerial as a result of non-covalent interactions between cross-linkingagents and affinity ligands on different conjugates. The conjugates alsoinclude a drug. The drug and affinity ligands may be covalently ornon-covalently bound to the conjugate framework. In general, thesematerials are designed so that an increase in the local concentration ofexogenous target molecule triggers the release of conjugates. Thedisclosure also provides methods of using these materials wherein atriggering amount of the exogenous target molecule is administered to apatient who has previously been administered a material of the presentdisclosure. The disclosure also provides methods of making thesematerials. In another aspect, the disclosure provides exemplarycross-linked materials and exogenous target molecules.

DEFINITIONS

Definitions of specific functional groups, chemical terms, and generalterms used throughout the specification are described in more detailbelow. For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 75^(th) Ed., inside cover,and specific functional groups are generally defined as describedtherein. Additionally, general principles of organic chemistry, as wellas specific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; Carruthers, SomeModern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Acyl—As used herein, the term “acyl,” refers to a group having thegeneral formula —C(═O)R^(X1), —C(═O)OR^(X1), —C(═O)—O—C(═O)R^(X1),—C(═O)SR^(X1), —C(═O)N(R^(X1))₂, —C(═S)R^(X1), —C(═S)N(R^(X1))₂, and—C(═S)S(R^(X1)), —C(═NR^(X1))R^(X1), —C(═NR^(X1))OR^(X1),—C(═NR^(X1))SR^(X1), and —C(═NR^(X1))N(R^(X1))₂, wherein R^(X1) ishydrogen; halogen; substituted or unsubstituted hydroxyl; substituted orunsubstituted thiol; substituted or unsubstituted amino; substituted orunsubstituted acyl; cyclic or acyclic, substituted or unsubstituted,branched or unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; cyclic oracyclic, substituted or unsubstituted, branched or unbranched alkyl;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedalkenyl; substituted or unsubstituted alkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- ordi-aliphaticamino, mono- or di-heteroaliphaticamino, mono- ordi-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, ormono- or di-heteroarylamino; or two R^(X1) groups taken together form a5- to 6-membered heterocyclic ring. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas. Acylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

Aliphatic—As used herein, the term “aliphatic” or “aliphatic group”denotes an optionally substituted hydrocarbon moiety that may bestraight-chain (i.e., unbranched), branched, or cyclic (“carbocyclic”)and may be completely saturated or may contain one or more units ofunsaturation, but which is not aromatic. Unless otherwise specified,aliphatic groups contain 1-12 carbon atoms. In some embodiments,aliphatic groups contain 1-6 carbon atoms. In some embodiments,aliphatic groups contain 1-4 carbon atoms, and in yet other embodimentsaliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groupsinclude, but are not limited to, linear or branched, alkyl, alkenyl, andalkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkenyl—As used herein, the term “alkenyl” denotes an optionallysubstituted monovalent group derived from a straight- or branched-chainaliphatic moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. In certain embodiments, the alkenylgroup employed in the invention contains 2-6 carbon atoms. In certainembodiments, the alkenyl group employed in the invention contains 2-5carbon atoms. In some embodiments, the alkenyl group employed in theinvention contains 2-4 carbon atoms. In another embodiment, the alkenylgroup employed contains 2-3 carbon atoms. Alkenyl groups include, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike.

Alkyl—As used herein, the term “alkyl” refers to optionally substitutedsaturated, straight- or branched-chain hydrocarbon radicals derived froman aliphatic moiety containing between 1-6 carbon atoms by removal of asingle hydrogen atom. In some embodiments, the alkyl group employed inthe invention contains 1-5 carbon atoms. In another embodiment, thealkyl group employed contains 1-4 carbon atoms. In still otherembodiments, the alkyl group contains 1-3 carbon atoms. In yet anotherembodiments, the alkyl group contains 1-2 carbons. Examples of alkylradicals include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl,tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl,n-decyl, n-undecyl, dodecyl, and the like.

Alkynyl—As used herein, the term “alkynyl” refers to an optionallysubstituted monovalent group derived from a straight- or branched-chainaliphatic moiety having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. In certain embodiments, the alkynylgroup employed in the invention contains 2-6 carbon atoms. In certainembodiments, the alkynyl group employed in the invention contains 2-5carbon atoms. In some embodiments, the alkynyl group employed in theinvention contains 2-4 carbon atoms. In another embodiment, the alkynylgroup employed contains 2-3 carbon atoms. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl (propargyl),1-propynyl, and the like.

Aryl—As used herein, the term “aryl” used alone or as part of a largermoiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to anoptionally substituted monocyclic and bicyclic ring systems having atotal of five to 10 ring members, wherein at least one ring in thesystem is aromatic and wherein each ring in the system contains three toseven ring members. The term “aryl” may be used interchangeably with theterm “aryl ring”. In certain embodiments of the present invention,“aryl” refers to an aromatic ring system which includes, but not limitedto, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bearone or more substituents.

Arylalkyl—As used herein, the term “arylalkyl” refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

Bivalent hydrocarbon chain—As used herein, the term “bivalenthydrocarbon chain” (also referred to as a “bivalent alkylene group”) isa polymethylene group, i.e., —(CH₂)_(z)—, wherein z is a positiveinteger from 1 to 30, from 1 to 20, from 1 to 12, from 1 to 8, from 1 to6, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 30, from 2 to 20,from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or from 2 to 3. Asubstituted bivalent hydrocarbon chain is a polymethylene group in whichone or more methylene hydrogen atoms are replaced with a substituent.Suitable substituents include those described below for a substitutedaliphatic group.

Carbonyl—As used herein, the term “carbonyl” refers to a monovalent orbivalent moiety containing a carbon-oxygen double bond. Non-limitingexamples of carbonyl groups include aldehydes, ketones, carboxylicacids, ester, amide, enones, acyl halides, anhydrides, ureas,carbamates, carbonates, thioesters, lactones, lactams, hydroxamates,isocyanates, and chloroformates.

Cycloaliphatic—As used herein, the terms “cycloaliphatic”, “carbocycle”,or “carbocyclic”, used alone or as part of a larger moiety, refer to anoptionally substituted saturated or partially unsaturated cyclicaliphatic monocyclic or bicyclic ring systems, as described herein,having from 3 to 10 members. Cycloaliphatic groups include, withoutlimitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl,cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloalkylhas 3-6 carbons.

Halogen—As used herein, the terms “halo” and “halogen” refer to an atomselected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine(bromo, —Br), and iodine (iodo, —I).

Heteroaliphatic—As used herein, the terms “heteroaliphatic” or“heteroaliphatic group”, denote an optionally substituted hydrocarbonmoiety having, in addition to carbon atoms, from one to fiveheteroatoms, that may be straight-chain (i.e., unbranched), branched, orcyclic (“heterocyclic”) and may be completely saturated or may containone or more units of unsaturation, but which is not aromatic. Unlessotherwise specified, heteroaliphatic groups contain 1-6 carbon atomswherein 1-3 carbon atoms are optionally and independently replaced withheteroatoms selected from oxygen, nitrogen and sulfur. In someembodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein1-2 carbon atoms are optionally and independently replaced withheteroatoms selected from oxygen, nitrogen and sulfur. In yet otherembodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1carbon atom is optionally and independently replaced with a heteroatomselected from oxygen, nitrogen and sulfur. Suitable heteroaliphaticgroups include, but are not limited to, linear or branched, heteroalkyl,heteroalkenyl, and heteroalkynyl groups.

Heteroaralkyl—As used herein, the term “heteroaralkyl” refers to analkyl group substituted by a heteroaryl, wherein the alkyl andheteroaryl portions independently are optionally substituted.

Heteroaryl—As used herein, the term “heteroaryl” used alone or as partof a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refersto an optionally substituted group having 5 to 10 ring atoms, preferably5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in acyclic array; and having, in addition to carbon atoms, from one to fiveheteroatoms. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, carbocyclic, orheterocyclic rings, where the radical or point of attachment is on theheteroaromatic ring. Non limiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, andtetrahydroisoquinolinyl. A heteroaryl group may be mono- or bicyclic.The term “heteroaryl” may be used interchangeably with the terms“heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of whichterms include rings that are optionally substituted.

Heteroatom—As used herein, the term “heteroatom” refers to nitrogen,oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur,and any quaternized form of a basic nitrogen. The term “nitrogen” alsoincludes a substituted nitrogen.

Heterocyclic—As used herein, the terms “heterocycle”, “heterocyclyl”,“heterocyclic radical”, and “heterocyclic ring” are used interchangeablyand refer to a stable optionally substituted 5- to 7-membered monocyclicor 7- to 10-membered bicyclic heterocyclic moiety that is eithersaturated or partially unsaturated, and having, in addition to carbonatoms, one or more heteroatoms, as defined above. A heterocyclic ringcan be attached to its pendant group at any heteroatom or carbon atomthat results in a stable structure and any of the ring atoms can beoptionally substituted. Examples of such saturated or partiallyunsaturated heterocyclic radicals include, without limitation,tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclicgroup”, “heterocyclic moiety”, and “heterocyclic radical”, are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or carbocyclic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

Unsaturated—As used herein, the term “unsaturated”, means that a moietyhas one or more double or triple bonds.

Partially unsaturated—As used herein, the term “partially unsaturated”refers to a ring moiety that includes at least one double or triplebond. The term “partially unsaturated” is intended to encompass ringshaving multiple sites of unsaturation, but is not intended to includearyl or heteroaryl moieties, as herein defined.

Optionally substituted—As described herein, compounds of the inventionmay contain “optionally substituted” moieties. In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat one or more hydrogens of the designated moiety are replaced with asuitable substituent. Unless otherwise indicated, an “optionallysubstituted” group may have a suitable substituent at each substitutableposition of the group, and when more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. Combinations of substituents envisioned bythis invention are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable”, as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘); —NO₂; —CN;—N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘)) S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substitutedas defined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur, or, notwithstanding the definition above, twoindependent occurrences of R^(∘), taken together with their interveningatom(s), form a 3-12 saturated, partially unsaturated, or aryl mono orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(•), -(haloR^(•)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(•), —(CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(•), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(•),—(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂ NH₂, —(CH₂)₀₋₂NHR^(•),—(CH₂)₀₋₂NR^(•) ₂, —NO₂, —SiR^(•) ₃, —OSiR^(•) ₃, —C(O)SR^(•), —(C₁₋₄straight or branched alkylene)C(O)OR^(•), or —SSR^(•) wherein each R^(•)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH,—C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN,—C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein eachR^(•) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable protecting group—As used herein, the term “suitable protectinggroup,” refers to amino protecting groups or hydroxyl protecting groupsdepending on its location within the compound and includes thosedescribed in detail in Protecting Groups in Organic Synthesis, T. W.Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999.

Suitable amino-protecting groups include methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine(Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Suitable hydroxyl protecting groups include methyl, methoxylmethyl(MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.

Agglutinated—When two or more cells are “agglutinated” by across-linking agent as described herein, they are each physicallyassociated with the cross-linking agent in a cell-agent-cell complex.Typically, agglutination only occurs once the cross-linking agentconcentration reaches a threshold concentration. This concentration isreferred to as the minimum agglutination concentration (MAC). The MACfor a given cross-linking agent is commonly measured using aspectrophotometric plate reader that can quantify changes in solutionabsorbance.

Aptamer—As used herein, the term “aptamer” refers to a polynucleotide orpolypeptide that binds specifically to a target molecule. In general, anaptamer is said to “bind specifically” to its target molecule if itassociates at a detectable level with the target molecule and does notassociate detectably with unrelated molecular entities (e.g., moleculeswhich share no common structural features with the target molecule)under similar conditions. Specific association between a target moleculeand an aptamer will typically be dependent upon the presence of aparticular structural feature of the target molecule such as an epitoperecognized by the aptamer. Generally, if an aptamer is specific forepitope A, the presence of a molecule containing epitope A or thepresence of free unlabeled epitope A in a reaction containing both freelabeled epitope A and the aptamer thereto, will reduce the amount oflabeled epitope A that binds to the aptamer. In general, it is to beunderstood that specificity need not be absolute. Indeed, it is wellknown in the art that aptamers may cross-react with other epitopes inaddition to the target epitope. Such cross-reactivity may be acceptabledepending upon the application for which the aptamer is to be used. Thusthe degree of specificity of an aptamer will depend on the context inwhich it is being used. It is also to be understood that specificity maybe evaluated in the context of additional factors such as the affinityof the aptamer for the target molecule versus the affinity of theaptamer for non-target molecules.

Associated—As used herein, two entities are physically “associated” withone another when they are bound by direct non-covalent interactions.Desirable non-covalent interactions include those of the type whichoccur between an immunoglobulin molecule and an antigen for which theimmunoglobulin is specific, for example, ionic interactions, hydrogenbonds, van der Waals interactions, hydrophobic interactions, etc. Thestrength, or affinity of the physical association can be expressed interms of the dissociation constant (K_(d)) of the interaction, wherein asmaller K_(d) represents a greater affinity. For example, theassociation properties of a selected cross-linking agent and targetmolecule can be quantified using methods well known in the art.

Biodegradable—As used herein, the term “biodegradable” refers tomolecules that degrade (i.e., lose at least some of their covalentstructure) under physiological or endosomal conditions. Biodegradablemolecules are not necessarily hydrolytically degradable and may requireenzymatic action to degrade.

Biomolecule—As used herein, the term “biomolecule” refers to molecules(e.g., polypeptides, amino acids, polynucleotides, nucleotides,polysaccharides, sugars, lipids, nucleoproteins, glycoproteins,lipoproteins, steroids, metabolites, etc.) whether naturally-occurringor artificially created (e.g., by synthetic or recombinant methods) thatare commonly found in cells and tissues. Specific classes ofbiomolecules include, but are not limited to, enzymes, receptors,neurotransmitters, hormones, cytokines, cell response modifiers such asgrowth factors and chemotactic factors, antibodies, vaccines, haptens,toxins, interferons, ribozymes, anti-sense agents, plasmids, DNA, andRNA.

Drug—As used herein, the term “drug” refers to small molecules orbiomolecules that alter, inhibit, activate, or otherwise affect abiological event. For example, drugs may include, but are not limitedto, anti-AIDS substances, anti-cancer substances, antibiotics,anti-diabetic substances, immunosuppressants, anti-viral substances,enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or anti-thromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, and imagingagents. A more complete listing of exemplary drugs suitable for use inthe present invention may be found in “Pharmaceutical Substances:Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel,Thieme Medical Publishing, 1999; the “Merck Index: An Encyclopedia ofChemicals, Drugs, and Biologicals”, edited by Susan Budavari et al., CRCPress, 1996, and the United States Pharmacopeia-25/Nationalformulary-20, published by the United States Pharmcopeial Convention,Inc., Rockville Md., 2001. Preferably, though not necessarily, the drugis one that has already been deemed safe and effective for use by theappropriate governmental agency or body. For example, drugs for humanuse listed by the FDA under 21 C.F.R. §§330.5, 331 through 361, and 440through 460; drugs for veterinary use listed by the FDA under 21 C.F.R.§§500 through 589, are all considered acceptable for use in accordancewith the present invention.

Exogenous—As used herein, an “exogenous” molecule is one which is notpresent at significant levels in a patient unless administered to thepatient. In certain embodiments the patient is a human. As used herein,a molecule is not present at significant levels in a patient if normalhuman serum includes less than 0.1 mM of the molecule. In certainembodiments normal human serum may include less than 0.08 mM, less than0.06 mM, or less than 0.04 mM of the molecule.

Hyperbranched—As used herein, a “hyperbranched” structure is a covalentstructure that includes at least one branched branch (e.g., adendrimeric structure). A hyperbranched structure may include polymericand/or non-polymeric substructures.

Normal human serum—As used herein, “normal human serum” is human serumobtained by pooling approximately equal amounts of the liquid portion ofcoagulated whole blood from eight or more healthy individuals. A healthyindividual is a randomly selected 18-30 year old who presents with nodisease symptoms at the time blood is drawn.

Percentage homology—As used herein, the terms “percentage homology”refer to the percentage of sequence identity between two sequences afteroptimal alignment as defined in the present disclosure. For example, twonucleotide sequences are said to be “identical” if the sequence ofnucleotides in the two sequences is the same when aligned for maximumcorrespondence as described below. Sequence comparisons between twonucleotide sequences are typically performed by comparing sequences oftwo optimally aligned sequences over a region or “comparison window” toidentify and compare regions of sequence similarity. Optimal alignmentof sequences for comparison may be conducted by the local homologyalgorithm of Smith and Waterman, Ad. App. Math. 2:482 (1981), by thehomology alignment algorithm of Neddleman and Wunsch, J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementation of these algorithms, or by visual inspection.

Percentage of sequence identity—“Percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over acomparison window, where the portion of the nucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleotide residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison and multiplying theresult by 100 to yield the percentage of sequence identity. Thisdefinition of sequence identity given above is the definition that wouldbe used by one of ordinary skill in the art. The definition by itselfdoes not need the help of any algorithm. The algorithms are only helpfulto facilitate the optimal alignments of sequences, rather than calculatesequence identity. From this definition, it follows that there is a welldefined and only one value for the sequence identity between twocompared sequences which value corresponds to the value obtained for theoptimal alignment.

Polymer—As used herein, a “polymer” or “polymeric structure” is astructure that includes a string of covalently bound monomers. A polymercan be made from one type of monomer or more than one type of monomer.The term “polymer” therefore encompasses copolymers, includingblock-copolymers in which different types of monomer are groupedseparately within the overall polymer. A polymer can be linear orbranched.

Polynucleotide—As used herein, a “polynucleotide” is a polymer ofnucleotides. The terms “polynucleotide”, “nucleic acid”, and“oligonucleotide” may be used interchangeably. The polymer may includenatural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine,uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine,dihydrouridine, methylpseudouridine, 1-methyl adenosine, 1-methylguanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modifiedbases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), or modifiedphosphate groups (e.g., phosphorothioates and 5′-N-phosphoramiditelinkages).

Polypeptide—As used herein, a “polypeptide” is a polymer of amino acids.The terms “polypeptide”, “protein”, “oligopeptide”, and “peptide” may beused interchangeably. Polypeptides may contain natural amino acids,non-natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain) and/or amino acidanalogs as are known in the art. Also, one or more of the amino acidresidues in a polypeptide may be modified, for example, by the additionof a chemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. Thesemodifications may include cyclization of the peptide, the incorporationof D-amino acids, etc.

Polysaccharide—As used herein, a “polysaccharide” is a polymer ofsaccharides. The terms “polysaccharide”, “carbohydrate”, and“oligosaccharide”, may be used interchangeably. The polymer may includenatural saccharides (e.g., arabinose, lyxose, ribose, xylose, ribulose,xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose,talose, fructose, psicose, sorbose, tagatose, mannoheptulose,sedoheptulose, octolose, and sialose) and/or modified saccharides (e.g.,2′-fluororibose, 2′-deoxyribose, and hexose). Exemplary disaccharidesinclude sucrose, lactose, maltose, trehalose, gentiobiose, isomaltose,kojibiose, laminaribiose, mannobiose, melibiose, nigerose, rutinose, andxylobiose.

Small molecule—As used herein, the term “small molecule” refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis), that have a relatively low molecular weight.Typically, small molecules are monomeric and have a molecular weight ofless than about 1500 g/mol.

Treat—As used herein, the term “treat” (or “treating”, “treated”,“treatment”, etc.) refers to the administration of a material of thepresent disclosure to a subject in need thereof with the purpose toalleviate, relieve, alter, ameliorate, improve or affect a condition(e.g., diabetes), a symptom or symptoms of a condition (e.g.,hyperglycemia), or the predisposition toward a condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the chemical structures of AEG, AEM, AEBM and AETM. Theaffinity of these sugar based affinity ligands for Con A increases asshown.

FIG. 2: shows the chemical structures of some exemplary conjugatesincluding the TSB-C4 based conjugate used in the examples.

FIG. 3: (a) Plot of (♦) serum insulin and (◯) blood glucose levelsfollowing subcutaneous injection in non-diabetic SD rats at time 0 withTSB-C4-AEBM-2-insulin/native Con A (an α-methyl mannose-responsivematerial). An i.p. injection of α-methyl mannose was administered at 120min as indicated by the *.

FIG. 4: is a schematic of a cross-linked material 10 which is capable ofcontrollably releasing conjugates 20 in response to an exogenous targetmolecule. The materials are prepared by combining the conjugates 20 withmultivalent cross-linking agents 30 that non-covalently bind theaffinity ligands 40 of the conjugates 20 and thereby cross-link theconjugates 20 to form the cross-linked material 10. The non-covalentbonds between the multivalent cross-linking agents 30 and the affinityligands 40 are competitively dissociated in the presence of excessamounts of the exogenous target molecule.

FIG. 5: shows the structure of wild-type human insulin.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

This application refers to a number of documents including patent andnon-patent documents. The entirety of each of these documents isincorporated herein by reference. In one aspect, the disclosure providescross-linked materials that include multivalent cross-linking agentsthat bind an exogenous molecule; and conjugates that include two or moreseparate affinity ligands bound to a conjugate framework, wherein thetwo or more affinity ligands compete with the exogenous molecule forbinding with the cross-linking agents and wherein conjugates arecross-linked within the material as a result of non-covalentinteractions between cross-linking agents and affinity ligands ondifferent conjugates. The conjugates also include a drug. The drug andaffinity ligands may be covalently or non-covalently bound to theconjugate framework. In general, these materials are designed so that anincrease in the local concentration of exogenous molecule triggers therelease of conjugates. The disclosure also provides methods of usingthese materials wherein a triggering amount of the exogenous targetmolecule is administered to a patient who has previously beenadministered a material of the present disclosure. The disclosure alsoprovides methods of making these materials. In another aspect, thedisclosure provides exemplary cross-linked materials and exogenoustarget molecules.

The cross-linking agents bind an exogenous molecule (e.g., withoutlimitation α-methyl-mannose, mannose, L-fucose, N-acetyl glucosamine, asynthetic drug such as morphine, etc.) and are multivalent. Theconjugates include a conjugate framework with two or more separateaffinity ligands that compete with the exogenous molecule for bindingwith the cross-linking agents. When cross-linking agents and conjugatesare combined in the absence of the exogenous molecule, a non-covalentlycross-linked material is formed. When the material is placed in thepresence of free exogenous molecules these compete for the interactionsbetween the cross-linking agents and the conjugates. Above a certainconcentration of free exogenous molecule, the level of competitionbecomes such that the material begins to degrade by releasingconjugates. As a result, conjugates are released from the material in amanner which is directly tied to the local concentration of theexogenous molecule. In various embodiments, the material releasessubstantially no conjugates in normal human serum. The latter propertyensures that there is substantially no uncontrolled release ofconjugates from the material in the absence of the exogenous molecule.As discussed below, in various embodiments it may be desirable to adjustthe properties of the material so that it does release amounts ofconjugate in normal human serum (e.g., to provide an endogenouslycontrolled component in addition to an externally triggered component).

Exogenous Target Molecule

The present disclosure is not limited to any particular exogenousmolecule. In the Examples we describe a material which is triggered byα-methyl-mannose. We chose this particular mannose derivative forpurposes of illustrating the invention because it has a much higheraffinity (about 40 fold) for the lectin concanavalin A (Con A) thanendogenous glucose. This difference in binding affinity enabled us touse a Con A cross-linked material which does not release conjugates inresponse to endogenous levels of glucose and yet releases conjugates inresponse to exogenous α-methyl-mannose (see Example 1). It will beappreciated that other exogenous glucose or mannose derivatives with ConA binding affinities that are similar to (or greater than)α-methyl-mannose could have been used as exogenous target molecules forthe material of Example 1. Without limitation these include, mannose,L-fucose, bimannose, methylbimannose, ethylbimmanose, trimannose,methyltrimannose, ethyltrimmanose, amino derivatives thereof, etc.Goldstein et al. provide a review of a number of Con A inhibitors andtheir relative affinities in J. Biol. Chem. 243: 2003-2007, 1968 andBiochemistry. 4: 876-883, 1965. Similarly, it is to be understood thatother exogenous saccharides (and derivatives thereof) could be used withcross-linking agents (e.g., other lectins, aptamers, etc.) thatrecognize saccharides other than glucose or mannose. In fact, in certainembodiments, it may be advantageous to use a cross-linking agent thatdoes not bind endogenous glucose. Exemplary lectins that do not bindglucose include those isolated from monocot plants such as Galanthusnivalis, Allium sativum, and Allium ursinum. As discussed below, onecould also use aptamers that have been selected for their lack ofglucose binding. Either of these approaches would reduce the risk ofrelease triggered by fluctuations in endogenous levels of glucose. Invarious embodiments, this approach can be extended so as to avoidrelease in the presence of other endogenous molecules, e.g., othermetabolites such as creatinine, urea, etc.

While the Examples and foregoing involve saccharide bindingcross-linking agents and exogenous saccharides it is to be understoodthat the invention is not limited to such systems. Indeed, while lectinbased systems will generally be limited to exogenous saccharides,aptamer based systems can be designed to bind many different exogenousmolecules. For example, the inventive methods can be used to produce across-linked material which releases drug conjugates that neutralize theeffects of an exogenous drug when the exogenous drug levels get toohigh, e.g., without limitation, a material that releases naltrexoneconjugates in response to high levels of an opioid such as morphine,etc. This latter example highlights the fact that, in variousembodiments, the exogenous molecule can be a molecule that is notintentionally administered to a patient. Thus, while the inventivematerials and methods are useful for situations where the trigger isadministered for the intentional purpose of releasing conjugates from apre-administered material, they may also be useful in situations wherethe material is present to counteract non-prescribed ingestion,injection or inhalation of an exogenous molecule (e.g., an opioid suchas morphine by a drug abuser).

Conjugates

The conjugates include two or more separate affinity ligands bound to aconjugate framework. The two or more separate affinity ligands competewith the exogenous target molecule for binding with the cross-linkingagent. The conjugates also include a drug. The affinity ligands and drugmay be covalently or non-covalently bound to the conjugate framework.

Affinity Ligands

The two or more separate affinity ligands may have the same or differentchemical structures. The two or more separate affinity ligands may havethe same chemical structure as the exogenous target molecule itself ormay be a chemically related species of the exogenous target molecule.The only requirement is that they compete with the exogenous targetmolecule for binding with the cross-linking agent. In certainembodiments, the relative affinity of the conjugate and exogenous targetmolecule for the cross-linking agent is in the range of 1:1 to 100:1(where a relative affinity of 100:1 means that, in an equilibriummixture of conjugate, exogenous target molecule and cross-linking agent(in pH 7 HEPES buffered saline at 37 C), the cross-linking agent willbind about equal molar amounts of conjugate and exogenous targetmolecule if the concentration of exogenous target molecule is 100× theconcentration of conjugate). In certain embodiments, the relativeaffinity is in the range of 1:1 to 50:1, 1:1 to 10:1, 1:1 to 5:1 or 1:1to 2:1. In various embodiments it may be advantageous for the affinityligands to have a different chemical structure from the exogenous targetmolecule, e.g., in order to fine tune the relative affinity of thecross-linking agent for the conjugates and the exogenous targetmolecule. For example, when the exogenous target molecule isα-methyl-mannose one might use a saccharide or a polysaccharide as oneor more of affinity ligands. Thus, in certain embodiments, the affinityligands are capable of competing with α-methyl-mannose for binding to alectin (e.g., without limitation Con A, mannan-binding lectin or MBL,etc.).

In certain embodiments, the affinity ligand is of formula (IVa) or(IVb):

wherein:each R¹ is independently hydrogen, —OR^(y), —N(R^(y))₂, —SR^(y), —O—Y,-G-Z, or —CH₂R^(x);each R^(x) is independently hydrogen, —OR^(y), —N(R^(y))₂, —SR^(y), or—O—Y;each R^(y) is independently —R², —SO₂R², —S(O)R², —P(O)(OR²)₂, —C(O)R²,—CO₂R², or —C(O)N(R²)₂;each Y is independently a monosaccharide, disaccharide, ortrisaccharide;

-   each G is independently a covalent bond or an optionally substituted    C₁₋₉ alkylene, wherein one or more methylene units of G is    optionally replaced by —O—, —S—, —N(R²)—, —C(O)—, —OC(O)—, —C(O)O—,    —C(O)N(R²)—, —N(R²)C(O)—, —N(R²)C(O)N(R²)—, —SO₂—, —SO₂N(R²)—,    —N(R²)SO₂—, or —N(R²)SO₂N(R²)—;-   each Z is independently halogen, —N(R²)₂, —OR², —SR², —N₃, —C≡CR²,    —CO₂R², —C(O)R², or —OSO₂R²; and-   each R² is independently hydrogen or an optionally substituted group    selected from C₁₋₆ aliphatic, phenyl, a 4-7 membered heterocyclic    ring having 1-2 heteroatoms selected from nitrogen, oxygen, or    sulfur, or a 5-6 membered monocyclic heteroaryl ring having 1-4    heteroatoms selected from nitrogen, oxygen, or sulfur.

In certain embodiments, the affinity ligand of formula (IVa) or (IVb) isa monosaccharide. In certain embodiments, the affinity ligand is adisaccharide. In certain embodiments, the affinity ligand is atrisaccharide. In certain embodiments, the affinity ligand is atetrasaccharide. In certain embodiments, the affinity ligand comprisesno more than four saccharide moieties.

As defined generally above, each R¹ is independently hydrogen, —OR^(y),—N(R^(y))₂, —SR^(y), —O—Y, -G-Z, or —CH₂R^(x). In certain embodiments,R¹ is hydrogen. In certain embodiments, R¹ is —OH. In other embodiments,R¹ is —NHC(O)CH₃. In certain embodiments, R¹ is —O—Y. In certain otherembodiments, R¹ is -G-Z. In some embodiments, R¹ is —CH₂OH. In otherembodiments, R¹ is —CH₂—O—Y. In yet other embodiments, R¹ is —NH₂. Oneof ordinary skill in the art will appreciate that each R¹ substituent informula (IVa) or (IVb) may be of (R) or (S) stereochemistry.

As defined generally above, each R^(x) is independently hydrogen,—OR^(y), —N(R^(y))₂, —SR^(y), or —O—Y. In some embodiments, R^(x) ishydrogen. In certain embodiments, R^(x) is —OH. In other embodiments,R^(x) is —O—Y.

As defined generally above, each R^(y) is independently —R², —SO₂R²,—S(O)R², —P(O)(OR²)₂, —C(O)R², —CO₂R₂, or —C(O)N(R²)₂. In someembodiments, R^(y) is hydrogen. In other embodiments, R^(y) is —R². Insome embodiments, R^(y) is —C(O)R². In certain embodiments, R^(y) isacetyl. In other embodiments, R^(y) is —SO₂R², —S(O)R², —P(O)(OR²)₂,—CO₂R², or —C(O)N(R²)₂.

As defined generally above, Y is a monosaccharide, disaccharide, ortrisaccharide. In certain embodiments, Y is a monosaccharide. In someembodiments, Y is a disaccharide. In other embodiments, Y is atrisaccharide. In some embodiments, Y is mannose, glucose, fructose,galactose, rhamnose, or xylopyranose. In some embodiments, Y is sucrose,maltose, turanose, trehalose, cellobiose, or lactose. In certainembodiments, Y is mannose. In certain embodiments, Y is D-mannose. Oneof ordinary skill in the art will appreciate that the saccharide Y isattached to the oxygen group of —O—Y through anomeric carbon to form aglycosidic bond. The glycosidic bond may be of an alpha or betaconfiguration.

As defined generally above, each G is independently a covalent bond oran optionally substituted C₁₋₉ alkylene, wherein one or more methyleneunits of G is optionally replaced by —O—, —S—, —N(R²)—, —C(O)—, —OC(O)—,—C(O)O—, —C(O)N(R²)—, —N(R²)C(O)—, —N(R²)C(O)N(R²)—, —SO₂—, —SO₂N(R²)—,—N(R²)SO₂—, or —N(R²)SO₂N(R²)—. In some embodiments, G is a covalentbond. In certain embodiments, G is —O—C₁₋₈ alkylene. In certainembodiments, G is —OCH₂CH₂—.

As defined generally above, each Z is independently halogen, —N(R²)₂,—OR², —SR², —N₃, —C≡CR², —CO₂R², —C(O)R², or —OSO₂R². In someembodiments, Z is a halogen or —OSO₂R². In other embodiments, Z is —N₃or —C≡CR². In certain embodiments, Z is —N(R²)₂, —OR², or —SR². Incertain embodiments, Z is —SH. In certain embodiments, Z is —NH₂. Incertain embodiments, -G-Z is —OCH₂CH₂NH₂.

In some embodiments, the R¹ substituent on the C1 carbon of formula(IVa) is -G-Z to give a compound of formula (IVa-i):

wherein R¹, G, and Z are as defined and described herein.

In some embodiments, the ligand is of formula (IVa-ii):

wherein R¹, R^(x), G, and Z are as defined and described herein.

For example, in certain embodiments, one might use an affinity ligandthat includes one or more of the following: glucose, sucrose, maltose,mannose, derivatives of these (e.g., glucosamine, mannosamine,methylglucose, methylmannose, ethylglucose, ethylmannose, etc.) and/orhigher order combinations of these (e.g., a bimannose, a linear and/orbranched trimannose, etc.). In certain embodiments, the affinity ligandincludes a monosaccharide. In certain embodiments, the affinity ligandincludes a disaccharide. In certain embodiments, the affinity ligandincludes a trisaccharide. In certain embodiments, the affinity ligandincludes a polysaccharide. In some embodiments, the affinity ligandincludes a saccharide and one or more amine groups. In some embodiments,the affinity ligand is aminoethylglucose (AEG). In some embodiments, theaffinity ligand is aminoethylmannose (AEM). In some embodiments, theaffinity ligand is aminoethylbimannose (AEBM). In some embodiments, theaffinity ligand is aminoethyltrimannose (AETM). In some embodiments, theaffinity ligand is β-aminoethyl-N-acetylglucosamine (AEGA). In someembodiments, the affinity ligand is aminoethylfucose (AEF). In otherembodiments, the affinity ligand is D-glucosamine (GA). In certainembodiments, a saccharide ligand is of the “D” configuration. In otherembodiments, a saccharide ligand is of the “L” configuration. Below weshow the structures of these exemplary affinity ligands. Other exemplaryaffinity ligands will be recognized by those skilled in the art.

In various embodiments, the affinity ligand is a polysaccharide,glycopeptide or glycolipid. In certain embodiments, the affinity ligandincludes from 2-10 saccharide moieties, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or10 moieties. The terminal and/or internal residues of thepolysaccharide, glycopeptide or glycolipid may be selected based on thesaccharide specificity of the lectin in question (e.g., see Goldstein etal., Biochem. Biophys. Acta 317:500-504, 1973 and Lis et al., Ann. Rev.Biochem. 55:35-67, 1986).

In various embodiments, the affinity ligands for a particularconjugate/cross-linking agent combination may be selected empirically.According to such embodiments one or more affinity ligands are screenedbased on their relative binding affinities for the cross-linking agentas compared to the exogenous target molecule (and possibly endogenousmolecules as discussed below). In certain embodiments a library ofsaccharides and/or polysaccharides are screened in this manner. Asuitable affinity ligand will exhibit a detectable level of competitionwith the exogenous target molecule but will not compete so strongly thatit prevents all binding between the cross-linking agent and theexogenous target molecule.

In various embodiments, the affinity ligand will be selected based onits ability to compete with endogenous molecules for the cross-linkingagent. In particular, in certain embodiments it will be advantageous toselect an affinity ligand that has a much higher affinity for thecross-linking agent than a potential competing endogenous molecule(e.g., glucose when the cross-linking agent is glucose binding) and yetcan be competitively unbound by a suitable exogenous target molecule(e.g., α-methyl-mannose). This will minimize the extent of release froma cross-linked material in the absence of exogenous target molecule. Forexample, in Examples 1-2 we describe an exemplary conjugate with twoAEBM affinity ligands that showed very little background release in thepresence of physiological levels of endogenous glucose and yet produceda significant response to exogenous α-methyl-mannose. In otherembodiments it may be desirable to select affinity ligands that allowfor a certain amount of glucose responsive release and yet can beexogenously triggered in order to artificially modulate the release.

Other exemplary exogenous target molecule/affinity ligand combinationswill be recognized by those skilled in the art. In general, an affinityligand can be generated for any exogenous target molecule using thetarget molecule itself and/or by generating derivatives of the targetmolecule (e.g., by making chemical and/or stereochemical modificationsto the target molecule and then screening the resulting derivative forits relative affinity to the cross-linking agent in question).

As discussed in more detail below, the affinity ligands may be naturallypresent within the framework of the conjugate (e.g., as part of apolymer backbone or as a side group of a monomer). Alternatively (oradditionally) affinity ligands may be artificially incorporated into theconjugate framework (e.g., in the form of a chemical group that issynthetically added to a conjugate framework). In certain embodiments, aconjugate may include a framework which comprises 5 or more, 10 or more,20 or more, 25 or more, 50 or more, or 100 or more affinity ligands. Incertain embodiments, a conjugate may include a framework which comprises2-5, 2-10, 2-20, 2-25, 2-50 or 2-100 affinity ligands. In certainembodiments, a conjugate may include a framework which comprises as fewas 2, 3 or 4 separate affinity ligands.

Methods for conjugating affinity ligands to a conjugate framework arediscussed in more detail below. In certain embodiments, when theaffinity ligands include a saccharide, the conjugation (whether director indirect) involves the C1, C2 or C6 position of the saccharide. Incertain embodiments, the conjugation involves the C1 position. The C1position is also referred to as the anomeric carbon and may be connectedto the conjugate framework in the alpha or beta conformation. In certainembodiments, the C1 position is configured as the alpha anomer. In otherembodiments, the C1 position is configured as the beta anomer.

Drug

As noted above, the conjugate also comprises a drug. It is to beunderstood that a conjugate can comprise any drug. A conjugate cancomprise more than one copy of the same drug and/or can comprise morethan one type of drug. The conjugates are not limited to any particulardrug and may include small molecule drugs or biomolecular drugs. Ingeneral, the drug(s) used will depend on the disease or disorder to betreated.

For example, without limitation, in various embodiments a conjugate cancomprise any one of the following drugs: diclofenac, nifedipine,rivastigmine, methylphenidate, fluoroxetine, rosiglitazone, prednison,prednisolone, codeine, ethylmorphine, dextromethorphan, noscapine,pentoxiverine, acetylcysteine, bromhexine, epinephrine, isoprenaline,orciprenaline, ephedrine, fenoterol, rimiterol, ipratropium,cholinetheophyllinate, proxiphylline, bechlomethasone, budesonide,deslanoside, digoxine, digitoxin, disopyramide, proscillaridin,chinidine, procainamide, mexiletin, flecainide, alprenolol,proproanolol, nadolol, pindolol, oxprenolol, labetalol, tirnolol,atenolol, pentaeritrityltetranitrate, isosorbiddinitrate,isosorbidmononitrate, niphedipin, phenylamine, verapamil, diltiazem,cyclandelar, nicotinylalcholhol, inositolnicotinate, alprostatdil,etilephrine, prenalterol, dobutamine, dopamine, dihydroergotamine,guanetidine, betanidine, methyldopa, reserpine, guanfacine,trimethaphan, hydralazine, dihydralazine, prazosine, diazoxid,captopril, nifedipine, enalapril, nitroprusside, bendroflumethiazide,hydrochlorthiazide, metychlothiazide, polythiazide, chlorthalidon,cinetazon, clopamide, mefruside, metholazone, bumetanide, ethacrynacide,spironolactone, amiloride, chlofibrate, nicotinic acid, nicheritrol,brompheniramine, cinnarizine, dexchlorpheniramine, clemastine,antazoline, cyproheptadine, proethazine, cimetidine, ranitidine,sucralfat, papaverine, moxaverine, atropin, butylscopolamin, emepron,glucopyrron, hyoscyamine, mepensolar, methylscopolamine,oxiphencyclimine, probanteline, terodilin, sennaglycosides,sagradaextract, dantron, bisachodyl, sodiumpicosulfat, etulos,diphenolxylate, loperamide, salazosulfapyridine, pyrvin, mebendazol,dimeticon, ferrofumarate, ferrosuccinate, ferritetrasemisodium,cyanochobalamine, folid acid heparin, heparin co-factor, diculmarole,warfarin, streptokinase, urokinase, factor VIII, factor IX, vitamin K,thiopeta, busulfan, chlorambucil, cyclophosphamid, melfalan, carmustin,mercatopurin, thioguanin, azathioprin, cytarabin, vinblastin,vinchristin, vindesin, procarbazine, dacarbazine, lomustin, estramustin,teniposide, etoposide, cisplatin, amsachrin, aminogluthetimid,phosphestrol, medroxiprogresterone, hydroxiprogesterone, megesterol,noretisteron, tamoxiphen, ciclosporin, sulfosomidine, bensylpenicillin,phenoxymethylpenicillin, dicloxacillin, cloxacillin, flucoxacillin,ampicillin, amoxicillin, pivampicillin, bacampicillin, piperacillin,meziocillin, mecillinam, pivmecillinam, cephalotin, cephalexin,cephradin, cephadroxil, cephaclor, cefuroxim, cefotaxim, ceftazidim,cefoxitin, aztreonam, imipenem, cilastatin, tetracycline, lymecycline,demeclocycline, metacycline, oxitetracycline, doxycycline,chloramphenicol, spiramycin, fusidic acid, lincomycin, clindamycin,spectinomycin, rifampicin, amphotericin B, griseofulvin, nystatin,vancomycin, metronidazole, tinidazole, trimethoprim, norfloxacin,salazosulfapyridin, aminosalyl, isoniazid, etambutol, nitrofurantoin,nalidixic acid, metanamine, chloroquin, hydroxichloroquin, tinidazol,ketokonazol, acyclovir, interferon idoxuridin, retinal, tiamin,dexpantenol, pyridoxin, folic acid, ascorbic acid, tokoferol,phytominadion, phenfluramin, corticotropin, tetracosactid, tyrotropin,somatotoprin, somatrem, vasopressin, lypressin, desmopressin, oxytocin,chloriongonadotropin, cortison, hydrocortisone, fluodrocortison,prednison, prednisolon, fluoximesteron, mesterolon, nandrolon,stanozolol, oximetolon, cyproteron, levotyroxin, liotyronin,propylthiouracil, carbimazol, tiamazol, dihydrotachysterol,alfacalcidol, calcitirol, insulin, tolbutamid, chlorpropamid, tolazamid,glipizid, glibenclamid, phenobarbital, methyprylon, pyrityidion,meprobamat, chlordiazepoxid, diazepam, nitrazepam, baclofen, oxazepam,dikaliumclorazepat, lorazepam, flunitrazepam, alprazolam, midazolam,hydroxizin, dantrolene, chlometiazol, propionmazine, alimemazine,chlorpromazine, levomepromazine, acetophenazine, fluphenazine,perphenazine, prochlorperazine, trifluoperazine, dixyrazine,thiodirazine, periciazin, chloprothixene, tizanidine, zaleplon,zuclopentizol, flupentizol, thithixen, haloperidol, trimipramin,opipramol, chlomipramin, desipramin, lofepramin, amitriptylin,nortriptylin, protriptylin, maptrotilin, caffeine, cinnarizine,cyclizine, dimenhydinate, meclozine, prometazine, thiethylperazine,metoclopramide, scopolamine, phenobarbital, phenytoine, ethosuximide,primidone, carbamazepine, chlonazepam, orphenadrine, atropine,bensatropine, biperiden, metixene, procylidine, levodopa, bromocriptin,amantadine, ambenon, pyridostigmine, synstigmine, disulfiram, morphine,codeine, pentazocine, buprenorphine, pethidine, phenoperidine,phentanyl, methadone, piritramide, dextropropoxyphene, ketobemidone,acetylsalicylic acid, celecoxib, phenazone, phenylbutazone,azapropazone, piroxicam, ergotamine, dihydroergotamine, cyproheptadine,pizitifen, flumedroxon, allopurinol, probenecid, sodiummaurothiomalateauronofin, penicillamine, estradiol, estradiolvalerianate, estriol,ethinylestradiol, dihydrogesteron, lynestrenol, medroxiprogresterone,noretisterone, cyclophenile, clomiphene, levonorgestrel, mestranol,ornidazol, tinidazol, ekonazol, chlotrimazol, natamycine, miconazole,sulbentin, methylergotamine, dinoprost, dinoproston, gemeprost,bromocriptine, phenylpropanolamine, sodiumchromoglicate, azetasolamide,dichlophenamide, betacarotene, naloxone, calciumfolinate, in particularclonidine, thephylline, dipyradamol, hydrochlothiazade, scopolamine,indomethacine, furosemide, potassium chloride, morphine, ibuprofen,salbutamol, terbutalin, calcitonin, etc. It is to be undersrtood thatthis list is intended to be exemplary and that any drug, whether knownor later discovered, may be used in a conjugate of the presentdisclosure.

In various embodiments, a conjugate may include a hormonal drug whichmay be peptidic or non-peptidic, e.g., adrenaline, noradrenaline,angiotensin, atriopeptin, aldosterone, dehydroepiandrosterone,androstenedione, testosterone, dihydrotestosterone, calcitonin,calcitriol, calcidiol, corticotropin, cortisol, dopamine, estradiol,estrone, estriol, erythropoietin, follicle-stimulating hormone, gastrin,ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone,growth hormone-releasing hormone, human chorionic gonadotropin,histamine, human placental lactogen, insulin, insulin-like growthfactor, inhibin, leptin, a leukotriene, lipotropin, melatonin, orexin,oxytocin, parathyroid hormone, progesterone, prolactin,prolactin-releasing hormone, a prostglandin, renin, serotonin, secretin,somatostatin, thrombopoietin, thyroid-stimulating hormone,thyrotropin-releasing hormone (or thyrotropin), thyrotropin-releasinghormone, thyroxine, triiodothyronine, vasopressin, etc. In certainembodiments, the hormone may be selected from glucagon, insulin,insulin-like growth factor, leptin, thyroid-stimulating hormone,thyrotropin-releasing hormone (or thyrotropin), thyrotropin-releasinghormone, thyroxine, and triiodothyronine. It is to be understood thatthis list is intended to be exemplary and that any hormonal drug,whether known or later discovered, may be used in a conjugate of thepresent disclosure.

In various embodiments, a conjugate may include a thyroid hormone.

In various embodiments, a conjugate may include an anti-diabetic drug(i.e., a drug which has a beneficial effect on patients suffering fromdiabetes).

In various embodiments, a conjugate may include an insulin molecule. By“an insulin molecule” we intend to encompass both wild-type and modifiedforms of insulin as long as they are bioactive (i.e., capable of causinga detectable reduction in glucose when administered in vivo). Wild-typeinsulin includes insulin from any species whether in purified, syntheticor recombinant form (e.g., human insulin, porcine insulin, bovineinsulin, rabbit insulin, sheep insulin, etc.). A number of these areavailable commercially, e.g., from Sigma-Aldrich (St. Louis, Mo.). Avariety of modified forms of insulin are known in the art (e.g. seeCrotty and Reynolds, Pediatr. Emerg. Care. 23:903-905, 2007 and Gerich,Am. J. Med. 113:308-16, 2002 and references cited therein). Modifiedforms of insulin may be chemically modified (e.g., by addition of achemical moiety such as a PEG group or a fatty acyl chain as describedbelow) and/or mutated (i.e., by addition, deletion or substitution ofone or more amino acids). In general, a bioactive mutant form of insulinwill typically differ from wild-type insulin by 1-10 (e.g., from 1-5 or1-2) amino acid substitutions, additions or deletions. The wild-typesequence of human insulin (A-chain and B-chain) is shown below and inFIG. 5.

A-Chain (SEQ ID NO: 1): GIVEQCCTSICSLYQLENYCN B-Chain (SEQ ID NO: 2):FVNQHLCGSHLVEALYLVCGERGFFYTPKT

Human insulin differs from rabbit, porcine, bovine, and sheep insulinonly in amino acids A8, A9, A10, and B30 (see table below).

Amino Acid Position Insulin A8 A9 A10 B30 human Thr Ser Ile Thr rabbitThr Ser Ile Ser porcine Thr Ser Ile Ala bovine Ala Ser Val Ala sheep AlaGly Val Ala

In various embodiments, an insulin molecule of the present disclosure ismutated at the B28 and/or B29 positions of the B-peptide sequence. Forexample, insulin lispro (HUMALOG®) is a rapid acting insulin mutant inwhich the penultimate lysine and proline residues on the C-terminal endof the B-peptide have been reversed (Lys^(B28)Pro^(B29)-human insulin).This modification blocks the formation of insulin multimers. Insulinaspart (NOVOLOG®) is another rapid acting insulin mutant in whichproline at position B28 has been substituted with aspartic acid(Asp^(B28)-human insulin). This mutant also prevents the formation ofmultimers. In some embodiments, mutation at positions B28 and/or B29 isaccompanied by one or more mutations elsewhere in the insulinpolypeptide. For example, insulin glulisine (APIDRA®) is yet anotherrapid acting insulin mutant in which aspartic acid at position B3 hasbeen replaced by a lysine residue and lysine at position B29 has beenreplaced with a glutamic acid residue (Lys^(B3)Glu^(B29)-human insulin).

In various embodiments, an insulin molecule of the present disclosurehas an isoelectric point that is shifted relative to human insulin. Insome embodiments, the shift in isoelectric point is achieved by addingone or more arginine residues to the N-terminus of the insulin A-peptideand/or the C-terminus of the insulin B-peptide. Examples of such insulinpolypeptides include Arg^(A0)-human insulin, Arg^(B31)Arg^(B32)-humaninsulin, Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,Arg^(A0)Arg^(B31)Arg^(B32)-human insulin, andArg^(A0)Gly^(A21)Arg^(B31)Arg^(B32)-human insulin. By way of furtherexample, insulin glargine (LANTUS®) is an exemplary long acting insulinmutant in which Asp^(A21) has been replaced by glycine, and two arginineresidues have been added to the C-terminus of the B-peptide. The effectof these changes is to shift the isoelectric point, producing a solutionthat is completely soluble at pH 4. Thus, in some embodiments, aninsulin molecule of the present disclosure comprises an A-peptidesequence wherein A21 is Gly and B-peptide sequence wherein B31 isArg-Arg. It is to be understood that the present disclosure encompassesall single and multiple combinations of these mutations and any othermutations that are described herein (e.g., Gly^(A21)-human insulin,Gly^(A21)Arg^(B31)-human insulin, Arg^(B31)Arg^(B32)-human insulin,Arg^(B31)-human insulin).

In various embodiments, an insulin molecule of the present disclosure istruncated. For example, in certain embodiments, a B-peptide sequence ofan insulin polypeptide of the present disclosure is missing B1, B2, B3,B26, B27, B28, B29 and/or B30. In certain embodiments, combinations ofresidues are missing from the B-peptide sequence of an insulinpolypeptide of the present disclosure. For example, the B-peptidesequence may be missing residues B(1-2), B(1-3), B(29-30), B(28-30),B(27-30) and/or B(26-30). In some embodiments, these deletions and/ortruncations apply to any of the aforementioned insulin molecules (e.g.,without limitation to produce des(B30)-insulin lispro, des(B30)-insulinaspart, des(B30)-insulin glulisine, des(B30)-insulin glargine, etc.).

In some embodiments, an insulin molecule contains additional amino acidresidues on the N- or C-terminus of the A or B-peptide sequences. Insome embodiments, one or more amino acid residues are located atpositions A0, A21, B0 and/or B31. In some embodiments, one or more aminoacid residues are located at position A0. In some embodiments, one ormore amino acid residues are located at position A21. In someembodiments, one or more amino acid residues are located at position B0.In some embodiments, one or more amino acid residues are located atposition B31. In certain embodiments, an insulin molecule does notinclude any additional amino acid residues at positions A0, A21, B0 orB31.

In certain embodiments, an insulin molecule of the present disclosure ismutated such that one or more amidated amino acids are replaced withacidic forms. For example, asparagine may be replaced with aspartic acidor glutamic acid. Likewise, glutamine may be replaced with aspartic acidor glutamic acid. In particular, Asn^(A18), Asn^(A21), or Asn^(B3), orany combination of those residues, may be replaced by aspartic acid orglutamic acid. Gln^(A15) or Gln^(B4), or both, may be replaced byaspartic acid or glutamic acid. In certain embodiments, an insulinmolecule has aspartic acid at position A21 or aspartic acid at positionB3, or both.

One skilled in the art will recognize that it is possible to mutate yetother amino acids in the insulin molecule while retaining biologicalactivity. For example, without limitation, the following modificationsare also widely accepted in the art: replacement of the histidineresidue of position B10 with aspartic acid (His^(B10)→Asp^(B10));replacement of the phenylalanine residue at position B1 with asparticacid (Phe^(B1)→Asp^(B1)); replacement of the threonine residue atposition B30 with alanine (Thr^(B3)→Ala^(B30)); replacement of thetyrosine residue at position B26 with alanine (Tyr^(B26)→Ala^(B26)); andreplacement of the serine residue at position B9 with aspartic acid(Ser^(B9)→Asp^(B9)).

In various embodiments, an insulin molecule of the present disclosurehas a protracted profile of action. Thus, in certain embodiments, aninsulin molecule of the present disclosure may be acylated with a fattyacid. That is, an amide bond is formed between an amino group on theinsulin molecule and the carboxylic acid group of the fatty acid. Theamino group may be the alpha-amino group of an N-terminal amino acid ofthe insulin molecule, or may be the epsilon-amino group of a lysineresidue of the insulin molecule. An insulin molecule of the presentdisclosure may be acylated at one or more of the three amino groups thatare present in wild-type insulin or may be acylated on lysine residuethat has been introduced into the wild-type sequence. In certainembodiments, an insulin molecule may be acylated at position B1. Incertain embodiments, an insulin molecule may be acylated at positionB29. In certain embodiments, the fatty acid is selected from myristicacid (C14), pentadecylic acid (C15), palmitic acid (C16), heptadecylicacid (C17) and stearic acid (C18). For example, insulin detemir(LEVEMIR®) is a long acting insulin mutant in which Thr^(B30) has beendeleted, and a C14 fatty acid chain (myristic acid) has been attached toLys^(B29).

In some embodiments, the N-terminus of the A-peptide, the N-terminus ofthe B-peptide, the epsilon-amino group of Lys at position B29 or anyother available amino group in an insulin molecule of the presentdisclosure is covalently linked to a fatty acid moiety of generalformula:

where R^(F) is hydrogen or a C₁₋₃₀ alkyl group. In some embodiments,R^(F) is a C₁₋₂₀ alkyl group, a C₃₋₁₉ alkyl group, a C₅₋₁₈ alkyl group,a C₆₋₁₇ alkyl group, a C₈₋₁₆ alkyl group, a C₁₀₋₁₅ alkyl group, or aC₁₂₋₁₄ alkyl group. In certain embodiments, the insulin polypeptide isconjugated to the moiety at the A1 position. In certain embodiments, theinsulin polypeptide is conjugated to the moiety at the B1 position. Incertain embodiments, the insulin polypeptide is conjugated to the moietyat the epsilon-amino group of Lys at position B29. In certainembodiments, position B28 of the insulin molecule is Lys and theepsilon-amino group of Lys^(B28) is conjugated to the fatty acid moiety.In certain embodiments, position B3 of the insulin molecule is Lys andthe epsilon-amino group of Lys^(B3) is conjugated to the fatty acidmoiety. In some embodiments, the fatty acid chain is 8-20 carbons long.In some embodiments, the fatty acid is octanoic acid (C8), nonanoic acid(C9), decanoic acid (C10), undecanoic acid (C11), dodecanoic acid (C12),or tridecanoic acid (C13). In certain embodiments, the fatty acid ismyristic acid (C14), pentadecanoic acid (C15), palmitic acid (C16),heptadecanoic acid (C17), stearic acid (C18), nonadecanoic acid (C19),or arachidic acid (C20). For example, insulin detemir (LEVEMIR®) is along acting insulin mutant in which Thr^(B30) has been deleted, and aC14 fatty acid chain (myristic acid) is attached to Lys^(B29).

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: Lys^(B28)Pro^(B29)-human insulin (insulinlispro), Asp^(B28)-human insulin (insulin aspart),Lys^(B3)Glu^(B29)-human insulin (insulin glulisine),Arg^(B31)Arg^(B32)-human insulin (insulin glargine),N^(εB29)-myristoyl-des(B30)-human insulin (insulin detemir),Ala^(B26)-human insulin, Asp^(B1)-human insulin, Arg^(A0)-human insulin,Asp^(B1)Glu^(B13)-human insulin, Gly^(A21)-human insulin,Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,Arg^(A0)Arg^(B31)Arg^(B32)-human insulin,Arg^(A0)Gly^(A21)Arg^(B31)Arg^(B32)-human insulin, des(B30)-humaninsulin, des(B27)-human insulin, des(B28-B30)-human insulin,des(B1)-human insulin, des(B1-B3)-human insulin. In certain embodiments,an insulin molecule of the present disclosure comprises the mutationsand/or chemical modifications of one of the following insulin molecules:N^(εB29)-palmitoyl-human insulin, N^(εB29)-myrisotyl-human insulin,N^(εB28)-palmitoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-myristoyl-Lys^(B28)Pro^(B29)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-palmitoyl-des(B30)-human insulin,N^(εB30)-myristoyl-Thr^(B29)Lys^(B30)-human insulin,N^(εB30)-palmitoyl-Thr^(B29)Lys^(B30)-human insulin,N^(εB29)-(N-palmitoyl-γ-glutamyl)-des(B30)-human insulin,N^(εB29)-(N-lithocolyl-γ-glutamyl)-des(B30)-human insulin,N^(εB29)-(ω-carboxyheptadecanoyl)-des(B30)-human insulin,N^(εB29)-(ω-carboxyheptadecanoyl)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-octanoyl-human insulin,N^(εB29)-myristoyl-Gly^(A21)Arg^(B31)Arg^(B31)-human insulin,N^(εB29)-myristoyl-Gly^(A21)Gln^(B3)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-myristoyl-Arg^(A0)Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-Arg^(A0)Gly^(A21)Gln^(B3)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-myristoyl-Arg^(A0)Gly^(A21)Asp^(B3)Arg^(B31)Arg^(B32)-humaninsulin, N^(εB29)-myristoyl-Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-myristoyl-Arg^(A0)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-octanoyl-Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-octanoyl-Gly^(A21)Gln^(B3)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-octanoyl-Arg^(A0)Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-octanoyl-Arg^(A0)Gly^(A21)Gln^(B3)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB29)-octanoyl-Arg^(B0)Gly^(A21)Asp^(B3)Arg^(B31)Arg^(B32)-humaninsulin, N^(εB29)-octanoyl-Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-octanoyl-Arg^(A0)Arg^(B31)Arg^(B32)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin polypeptides:N^(εB28)-myristoyl-Gly^(A21)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-myristoyl-Gly^(A21)Gln^(B3)Lys^(B28)Pro^(B30)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-myristoyl-Arg^(A0)Gly^(A21)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-myristoyl-Arg^(A0)Gly^(A21)Gln^(B3)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-myristoyl-Arg^(A0)Gly^(A21)Asp^(B3)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin, N^(εB28)-myristoyl-Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin, N^(εB28)-myristoyl-arg^(A0)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-human insulin,N^(εB28)-octanoyl-Gly^(A21)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules:N^(εB28)-octanoyl-Gly^(A21)Gln^(B3)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-octanoyl-Arg^(A0)Gly^(A21)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-octanoyl-Arg^(A0)Gly^(A21)Gln^(B3)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-octanoyl-Arg^(A0)Gly^(A21)Asp^(B3)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin, N^(εB28)-octanoyl-Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB28)-octanoyl-Arg^(A0)Lys^(B28)Pro^(B29)Arg^(B31)Arg^(B32)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-tridecanoyl-des(B30)-humaninsulin, N^(εB29)-tetradecanoyl-des(B30)-human insulin,N^(εB29)-decanoyl-des(B30)-human insulin,N^(εB29)-dodecanoyl-des(B30)-human insulin,N^(εB29)-tridecanoyl-Gly^(A21)-des(B30)-human insulin,N^(εB29)-tetradecanoyl-Gly^(A21)-des(B30)-human insulin,N^(εB29)-decanoyl-Gly^(A21)-des(B30)-human insulin,N^(εB29)-dodecanoyl-Gly^(A21)-des(B30)-human insulin,N^(εB29)-tridecanoyl-Gly^(A21)Gln^(B3)-des(B30)-human insulin,N^(εB29)-tetradecanoyl-Gly^(A21)Gln^(B3)-des(B30)-human insulin,N^(εB29)-decanoyl-Gly^(A21)-Gln^(B3)-des(B30)-human insulin,N^(εB29)-dodecanoyl-Gly^(A21)-Gln^(B3)-des(B30)-human insulin,N^(εB29)-tridecanoyl-Ala^(A21)-des(B30)-human insulin,N^(εB29)-tetradecanoyl-Ala^(A21)-des(B30)-human insulin,N^(εB29)-decanoyl-Ala^(A21)-des(B30)-human insulin,N^(εB29)-dodecanoyl-Ala^(A21)-des(B30)-human insulin,N^(εB29)-tridecanoyl-Ala^(A21)-Gln^(B3)-des(B30)-human insulin,N^(εB29)-tetradecanoyl-Ala^(A21)Gln^(B3)-des(B30)-human insulin,N^(εB29)-decanoyl-Ala^(A21)Gln^(B3)-des(B30)-human insulin,N^(εB29)-dodecanoyl-Ala^(A21)Gln^(B3)-des(B30)-human insulin,N^(εB29)-tridecanoyl-Gln^(B3)-des(B30)-human insulin,N^(εB29)-tetradecanoyl-Gln^(B3)-des(B30)-human insulin,N^(εB29)-decanoyl-Gln^(B3)-des(B30)-human insulin,N^(εB29)-dodecanoyl-Gln^(B3)-des(B30)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-tridecanoyl-Gly^(A21)-humaninsulin, N^(εB29)-tetradecanoyl-Gly^(A21)-human insulin,N^(εB29)-decanoyl-Gly^(A21)-human insulin,N^(εB29)-dodecanoyl-Gly^(A21)-human insulin,N^(εB29)-tridecanoyl-Ala^(A21)-human insulin,N^(εB29)-tetradecanoyl-Ala^(A21)-human insulin,N^(εB29)-decanoyl-Ala^(A21)-human insulin,N^(εB29)-dodecanoyl-Ala^(A21)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules:N^(εB29)-tridecanoyl-Gly^(A21)Gln^(B3)-human insulin,N^(εB29)-tetradecanoyl-Gly^(A21)Gln^(B3)-human insulin,N^(εB29)-decanoyl-Gly^(A21)Gln^(B3)-human insulin,N^(εB29)-dodecanoyl-Gly^(A21)Gln^(B3)-human insulin,N^(εB29)-tridecanoyl-Ala^(A21)Gln^(B3)-human insulin,N^(εB29)-tetradecanoyl-Ala^(A21)Gln^(B3)-human insulin,N^(εB29)-decanoyl-Ala^(A21)Gln^(B3)-human insulin,N^(εB29)-dodecanoyl-Ala^(A21)Gln^(B3)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-tridecanoyl-Gln^(B3)-humaninsulin, N^(εB29)-tetradecanoyl-Gln^(B3)-human insulin,N^(εB29)-decanoyl-Gln^(B3)-human insulin,N^(εB29)-dodecanoyl-Gln^(B3)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-tridecanoyl-Glu^(B30)-humaninsulin, N^(εB29)-tetradecanoyl-Glu^(B30)-human insulin,N^(εB29)-decanoyl-Glu^(B30)-human insulin,N^(εB29)-dodecanoyl-Glu^(B30)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-tridecanoyl-Gly^(A21)Glu³⁰-humaninsulin, N^(εB29)-tetradecanoyl-Gly^(A21)Glu^(B30)-human insulin,N^(εB29)-decanoyl-Gly^(A21)Glu^(B30)-human insulin,N^(εB29)-dodecanoyl-Gly^(A21)Glu^(B30)-human insulin. In certainembodiments, an insulin molecule of the present disclosure comprises themutations and/or chemical modifications of one of the following insulinmolecules: N^(εB29)-tridecanoyl-Gly^(A21)Gln^(B3)Glu^(B30)-humaninsulin, N^(εB29)-tetradecanoyl-Gly^(A21)Gln^(B3)Glu^(B30)-humaninsulin, N^(εB29)-decanoyl-Gly^(A21)Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-dodecanoyl-Gly^(A21)Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-tridecanoyl-Ala^(A21)Glu^(B30)-human insulin,N^(εB29)-tetradecanoyl-Ala^(A21)Glu^(B30)-human insulin,N^(εB29)-decanoyl-Ala^(A21)Glu^(B30)-human insulin,N^(εB29)-dodecanoyl-Ala^(A21)Glu^(B30)-human insulin,N^(εB29)-tridecanoyl-Ala^(A21)Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-tetradecanoyl-Ala^(A21)Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-decanoyl-Ala^(A21)Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-dodecanoyl-Ala^(A21)Gln^(B3)Glu^(B30)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules:N^(εB29)-tridecanoyl-Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-tetradecanoyl-Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-decanoyl-Gln^(B3)Glu^(B30)-human insulin,N^(εB29)-dodecanoyl-Gln^(B3)Glu^(B30)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-formyl-human insulin,N^(αB1)-formyl-human insulin, N^(αA1)-formyl-human insulin,N^(εB29)-formyl-N^(αB1)-formyl-human insulin,N^(εB29)-formyl-N^(αA1)-formyl-human insulin,N^(αA1)-formyl-N^(αB1)-formyl-human insulin,N^(εB29)-formyl-N^(αA1)-formyl-N^(αB1)-formyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-acetyl-human insulin,N^(αB1)-acetyl-human insulin, N^(αA1)-acetyl-human insulin,N^(εB29)-acetyl-N^(αB1)-acetyl-human insulin,N^(εB29)-acetyl-N^(αA1)-acetyl-human insulin,N^(αA1)-acetyl-N^(αB1)-acetyl-human insulin,N^(εB29)-acetyl-N^(αA1)-acetyl-N^(αB1)-acetyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-propionyl-human insulin,N^(αB1)-propionyl-human insulin, N^(αA1)-propionyl-human insulin,N^(εB29)-acetyl-N^(αB1)-propionyl-human insulin,N^(εB29)-propionyl-N^(αA1)-propionyl-human insulin,N^(αA1)-propionyl-N^(αB1)-propionyl-human insulin,N^(εB29)-propionyl-N^(αA1)-propionyl-N^(αB1)-propionyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-butyryl-human insulin,N^(αB1)-butyryl-human insulin, N^(αA1)-butyryl-human insulin,N^(εB29)-butyryl-N^(αB1)-butyryl-human insulin,N^(εB29)-butyryl-N^(αA1)-butyryl-human insulin,N^(αA1)-butyryl-N^(αB1)-butyryl-human insulin,N^(εB29)-butyryl-N^(αA1)-butyryl-N^(αB1)-butyryl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-pentanoyl-human insulin,N^(αB1)-pentanoyl-human insulin, N^(αA1)-pentanoyl-human insulin,N^(εB29)-pentanoyl-N^(αB1)-pentanoyl-human insulin,N^(εB29)-pentanoyl-N^(αA1)-pentanoyl-human insulin,N^(αA1)-pentanoyl-N^(αB1)-pentanoyl-human insulin,N^(εB29)-pentanoyl-N^(αA1)-pentanoyl-N^(αB1)-pentanoyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-hexanoyl-human insulin,N^(αB1)-hexanoyl-human insulin, N^(αA1)-hexanoyl-human insulin,N^(εB29)-hexanoyl-N^(αB1)-hexanoyl-human insulin,N^(εB29)-hexanoyl-N^(αA1)-hexanoyl-human insulin,N^(αA1)-hexanoyl-N^(αB1)-hexanoyl-human insulin,N^(εB29)-hexanoyl-N^(αA1)-hexanoyl-N^(αB1)-hexanoyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-heptanoyl-human insulin,N^(αB1)-heptanoyl-human insulin, N^(αA1)-heptanoyl-human insulin,N^(εB29)-heptanoyl-N^(αB1)-heptanoyl-human insulin,N^(εB29)-heptanoyl-N^(αA1)-heptanoyl-human insulin,N^(αA1)-heptanoyl-N^(αB1)-heptanoyl-human insulin,N^(εB29)-heptanoyl-N^(αA1)-heptanoyl-N^(αB1)-heptanoyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(αB1)-octanoyl-human insulin,N^(αA1)-octanoyl-human insulin, N^(εB29)-octanoyl-N^(αB1)-octanoyl-humaninsulin, N^(εB29)-octanoyl-N^(αA1)-octanoyl-human insulin,N^(αA1)-octanoyl-N^(αB1)-octanoyl-human insulin,N^(εB29)-octanoyl-N^(αA1)-octanoyl-N^(αB1)-octanoyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-nonanoyl-human insulin,N^(αB1)-nonanoyl-human insulin, N^(αA1)-nonanoyl-human insulin,N^(εB29)-nonanoyl-N^(αB1)-nonanoyl-human insulin,N^(εB29)-nonanoyl-N^(αA1)-nonanoyl-human insulin,N^(αA1)-nonanoyl-N^(αB1)-nonanoyl-human insulin,N^(εB29)-nonanoyl-N^(αA1)-nonanoyl-N^(αB1)-nonanoyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB29)-decanoyl-human insulin,N^(αB1)-decanoyl-human insulin, N^(αA1)-decanoyl-human insulin,N^(εB29)-decanoyl-N^(αB1)-decanoyl-human insulin,N^(εB29)-decanoyl-N^(αA1)-decanoyl-human insulin,N^(αA1)-decanoyl-N^(αB1)-decanoyl-human insulin,N^(εB29)-decanoyl-N^(αA1)-decanoyl-N^(αB1)-decanoyl-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-formyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-formyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-formyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-formyl-N^(αB1)-formyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-formyl-N^(αA1)-formyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-formyl-N^(αB1)-formyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-formyl-N^(αA1)-formyl-N^(αB1)-formyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(εB29)-acetyl-Lys^(B28)Pro^(B29)-human insulin,N^(αB1)-acetyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-acetyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-acetyl-N^(αB1)-acetyl-Lys^(B28)Pro^(B29)-human insulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules:N^(εB28)-acetyl-N^(αA1)-acetyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-acetyl-N^(αB1)-acetyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-acetyl-N^(αA1)-acetyl-N^(αB1)-acetyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-propionyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-propionyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-propionyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-propionyl-N^(αB1)-propionyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-propionyl-N^(αA1)-propionyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-propionyl-N^(αB1)-propionyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-propionyl-N^(αA1)-propionyl-N^(αB1)-propionyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-butyryl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-butyryl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-butyryl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-butyryl-N^(αB1)-butyryl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-butyryl-N^(αA1)-butyryl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-butyryl-N^(αB1)-butyryl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-butyryl-N^(αA1)-butyryl-N^(αB1)-butyryl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-pentanoyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-pentanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-pentanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-pentanoyl-N^(αB1)-pentanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-pentanoyl-N^(αA1)-pentanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-pentanoyl-N^(αB1)-pentanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-pentanoyl-N^(αA1)-pentanoyl-N^(αB1)-pentanoyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-hexanoyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-hexanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-hexanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-hexanoyl-N^(αB1)-hexanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-hexanoyl-N^(αA1)-hexanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-hexanoyl-N^(αB1)-hexanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-hexanoyl-N^(αA1)-hexanoyl-N^(αB1)-hexanoyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-heptanoyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-heptanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-heptanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-heptanoyl-N^(αB1)-heptanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-heptanoyl-N^(αA1)-heptanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-heptanoyl-N^(αB1)-heptanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-heptanoyl-N^(αA1)-heptanoyl-N^(αB1)-heptanoyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-octanoyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-octanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-octanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-octanoyl-N^(αB1)-octanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-octanoyl-N^(αA1)-octanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-octanoyl-N^(αB1)-octanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-octanoyl-N^(αA1)-octanoyl-N^(αB1)-octanoyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-nonanoyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-nonanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-nonanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-nonanoyl-N^(αB1)-nonanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-nonanoyl-N^(αA1)-nonanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-nonanoyl-N^(αB1)-nonanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-nonanoyl-N^(αA1)-nonanoyl-N^(αB1)-nonanoyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules: N^(εB28)-decanoyl-Lys^(B28)Pro^(B29)-humaninsulin, N^(αB1)-decanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-decanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-decanoyl-N^(αB1)-decanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-decanoyl-N^(αA1)-decanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(αA1)-decanoyl-N^(αB1)-decanoyl-Lys^(B28)Pro^(B29)-human insulin,N^(εB28)-decanoyl-N^(αA1)-decanoyl-N^(αB1)-decanoyl-Lys^(B28)Pro^(B29)-humaninsulin.

In certain embodiments, an insulin molecule of the present disclosurecomprises the mutations and/or chemical modifications of one of thefollowing insulin molecules:N^(εB29)-pentanoyl-Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,N^(αB1)-hexanoyl-Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,N^(αA1)-heptanoyl-Gly^(A21)Arg^(B31)Arg^(B32)-human insulin,N^(εB29)-Octanoyl-N^(αB1)-octanoyl-Gly^(A21)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB29)-propionyl-N^(αA1)-propionyl-Gly^(A21)Arg^(B31)Arg^(B32)-humaninsulin, N^(αA1)-acetyl-N^(αB1)-acetyl-Gly^(A21)Arg^(B31)Arg^(B32)-humaninsulin,N^(εB29)-formyl-N^(αA1)-formyl-N^(αB1)-formyl-Gly^(A21)Arg^(B31)Arg^(B32)-humaninsulin, N^(εB29)-formyl-des(B26)-human insulin,N^(αB1)-acetyl-Asp^(B28)-human insulin,N^(εB29)-propionyl-N^(αA1)-propionyl-N^(αB1)-propionyl-Asp^(B1)Asp^(B3)AspB²¹-humaninsulin, N^(εB29)-pentanoyl-Gly^(A21)-human insulin,N^(αB1)-hexanoyl-Gly^(A21)-human insulin,N^(αA1)-heptanoyl-Gly^(A21)-human insulin,N^(εB29)-octanoyl-N^(αB1)-octanoyl-Gly^(A21)-human insulin,N^(εB29)-propionyl-N^(αA1)-propionyl-Gly^(A21)-human insulin,N^(αA1)-acetyl-N^(αB1)-acetyl-Gly^(A21)-human insulin,N^(εB29)-formyl-N^(αA1)-formyl-N^(αB1)-formyl-Gly^(A21)-human insulin,N^(εB29)-butyryl-des(B30)-human insulin, N^(αB1)-butyryl-des(B30)-humaninsulin, N^(αA1)-butyryl-des(B30)-human insulin,N^(εB29)-butyryl-N^(αB1)-butyryl-des(B30)-human insulin,N^(εB29)-butyryl-N^(αA1)-butyryl-des(B30)-human insulin,N^(αA1)-butyryl-N^(αB1)-butyryl-des(B30)-human insulin,N^(εB29)-butyryl-N^(αA1)-butyryl-N^(αB1)-butyryl-des(B30)-human insulin.

The present disclosure also encompasses modified forms of non-humaninsulins (e.g., porcine insulin, bovine insulin, rabbit insulin, sheepinsulin, etc.) that comprise any one of the aforementioned mutationsand/or chemical modifications.

These and other modified insulin molecules are described in detail inU.S. Pat. Nos. 6,906,028; 6,551,992; 6,465,426; 6,444,641; 6,335,316;6,268,335; 6,051,551; 6,034,054; 5,952,297; 5,922,675; 5,747,642;5,693,609; 5,650,486; 5,547,929; 5,504,188; 5,474,978; 5,461,031; and4,421,685; and in U.S. Pat. Nos. 7,387,996; 6,869,930; 6,174,856;6,011,007; 5,866,538; and 5,750,497, the entire disclosures of which arehereby incorporated by reference.

In various embodiments, an insulin molecule of the present disclosureincludes the three wild-type disulfide bridges (i.e., one betweenposition 7 of the A-chain and position 7 of the B-chain, a secondbetween position 20 of the A-chain and position 19 of the B-chain, and athird between positions 6 and 11 of the A-chain).

Methods for conjugating drugs including insulin molecules are describedbelow. In certain embodiments, an insulin molecule is conjugated to theconjugate framework via the A1 amino acid residue. In certainembodiments the A1 amino acid residue is glycine. It is to be understoodhowever, that the present disclosure is not limited to N-terminalconjugation and that in certain embodiments an insulin molecule may beconjugated via a non-terminal A-chain amino acid residue. In particular,the present disclosure encompasses conjugation via the epsilon-aminegroup of a lysine residue present at any position in the A-chain(wild-type or introduced by site-directed mutagenesis). It will beappreciated that different conjugation positions on the A-chain may leadto different reductions in insulin activity. In certain embodiments, aninsulin molecule is conjugated to the conjugate framework via the B1amino acid residue. In certain embodiments the B1 amino acid residue isphenylalanine. It is to be understood however, that the presentdisclosure is not limited to N-terminal conjugation and that in certainembodiments an insulin molecule may be conjugated via a non-terminalB-chain amino acid residue. In particular, the present disclosureencompasses conjugation via the epsilon-amine group of a lysine residuepresent at any position in the B-chain (wild-type or introduced bysite-directed mutagenesis). For example, in certain embodiments aninsulin molecule may be conjugated via the B29 lysine residue. In thecase of insulin glulisine, conjugation to the conjugate framework viathe B3 lysine residue may be employed. It will be appreciated thatdifferent conjugation positions on the B-chain may lead to differentreductions in insulin activity.

In certain embodiments, the ligands are conjugated to more than oneconjugation point on a drug such as an insulin molecule. For example, aninsulin molecule can be conjugated at both the A1 N-terminus and the B29lysine. In some embodiments, amide conjugation takes place in carbonatebuffer to conjugate at the B29 and A1 positions, but not at the B1position. In other embodiments, an insulin molecule can be conjugated atthe A1 N-terminus, the B1 N-terminus, and the B29 lysine. In yet otherembodiments, protecting groups are used such that conjugation takesplace at the B1 and B29 or B1 and A1 positions. It will be appreciatedthat any combination of conjugation points on an insulin molecule may beemployed. In some embodiments, at least one of the conjugation points isa mutated lysine residue, e.g., Lys^(A3).

In various embodiments, a conjugate may include an insulin sensitizer(i.e., a drug which potentiates the action of insulin). Drugs whichpotentiate the effects of insulin include biguanides (e.g., metformin)and glitazones. The first glitazone drug was troglitazone which turnedout to have severe side effects. Second generation glitazones includepioglitazone and rosiglitazone which are better tolerated althoughrosiglitazone has been associated with adverse cardiovascular events incertain trials.

In various embodiments, a conjugate may include an insulin secretagogue(i.e., a drug which stimulates insulin secretion by beta cells of thepancreas). For example, in various embodiments, a conjugate may includea sulfonylurea. Sulfonylureas stimulate insulin secretion by beta cellsof the pancreas by sensitizing them to the action of glucose.Sulfonylureas can, moreover, inhibit glucagon secretion and sensitizetarget tissues to the action of insulin. First generation sulfonylureasinclude tolbutamide, chlorpropamide and carbutamide. Second generationsulfonylureas which are active at lower doses include glipizide,glibenclamide, gliclazide, glibornuride and glimepiride. In variousembodiments, a conjugate may include a meglitinide. Suitablemeglitinides include nateglinide, mitiglinide and repaglinide. Theirhypoglycemic action is faster and shorter than that of sulfonylureas.Other insulin secretagogues include glucagon-like peptide 1 (GLP-1) andGLP-1 analogs (i.e., a peptide with GLP-1 like bioactivity that differsfrom GLP-1 by 1-10 amino acid substitutions, additions or deletionsand/or by a chemical modification). GLP-1 reduces food intake byinhibiting gastric emptying, increasing satiety through central actionsand by suppressing glucagon release. GLP-1 lowers plasma glucose levelsby increasing pancreas islet cell proliferation and increases insulinproduction following food consumption. GLP-1 may be chemically modified,e.g., by lipid conjugation as in liraglutide to extend its in vivohalf-life. Yet other insulin secretagogues include exendin-4 andexendin-4 analogs (i.e., a peptide with exendin-4 like bioactivity thatdiffers from exendin-4 by 1-10 amino acid substitutions, additions ordeletions and/or by a chemical modification). Exendin-4, found in thevenom of the Gila Monster, exhibits GLP-1 like bioactivity. It has amuch longer half-life than GLP-1 and, unlike GLP-1, it can be truncatedby 8 amino acid residues at its N-terminus without losing bioactivity.The N-terminal region of GLP-1 and exendin-4 are almost identical, asignificant difference being the second amino acid residue, alanine inGLP-1 and glycine in exendin-4, which gives exendin-4 its resistance toin vivo digestion. Exendin-4 also has an extra 9 amino acid residues atits C-terminus as compared to GLP-1. Mann et al. Biochem. Soc. Trans.35:713-716, 2007 and Runge et al., Biochemistry 46:5830-5840, 2007describe a variety of GLP-1 and exendin-4 analogs which may be used in aconjugate of the present disclosure. The short half-life of GLP-1results from enzymatic digestion by dipeptidyl peptidase IV (DPP-IV). Incertain embodiments, the effects of endogenous GLP-1 may be enhanced byadministration of a DPP-IV inhibitor (e.g., vildagliptin, sitagliptin,saxagliptin, linagliptin or alogliptin).

In various embodiments, a conjugate may include amylin or an amylinanalog (i.e., a peptide with amylin like bioactivity that differs fromamylin by 1-10 amino acid substitutions, additions or deletions and/orby a chemical modification). Amylin plays an important role in glucoseregulation (e.g., see Edelman and Weyer, Diabetes Technol. Ther.4:175-189, 2002). Amylin is a neuroendocrine hormone that is co-secretedwith insulin by the beta cells of the pancreas in response to foodintake. While insulin works to regulate glucose disappearance from thebloodstream, amylin works to help regulate glucose appearance in thebloodstream from the stomach and liver. Pramlintide acetate (SYMLIN®) isan exemplary amylin analog. Since native human amylin is amyloidogenic,the strategy for designing pramlintide involved substituting certainresidues with those from rat amylin, which is not amyloidogenic. Inparticular, proline residues are known to be structure-breakingresidues, so these were directly grafted from the rat sequence into thehuman sequence. Glu-10 was also substituted with an asparagine.

In various embodiments, a pre-conjugated drug may contain one or morereactive moieties (e.g., carboxyl or reactive ester, amine, hydroxyl,aldehyde, sulfhydryl, maleimidyl, alkynyl, azido, etc. moieties). Asdiscussed below, these reactive moieties may, in certain embodiments,facilitate the conjugation process. Specific examples include peptidicdrugs bearing alpha-terminal amine and/or epsilon-amine lysine groups.It will be appreciated that any of these reactive moieties may beartificially added to a known drug if not already present. For example,in the case of peptidic drugs a suitable amino acid (e.g., a lysine) maybe added or substituted into the amino acid sequence. In addition, asdiscussed in more detail below, it will be appreciated that theconjugation process may be controlled by selectively blocking certainreactive moieties prior to conjugation.

As discussed above, the present disclosure is not limited to anyparticular combination of drug and exogenous target molecule.

Conjugate Framework

Conjugates can be prepared from frameworks that naturally includeaffinity ligands (e.g., polysaccharides such as glycogen and dextrannaturally include glucose affinity ligands) and/or by artificiallyincorporating affinity ligands into a natural or synthetic framework. Itis to be understood that the conjugates of the present disclosure arenot limited to a particular framework. For example, conjugates may beprepared using frameworks that include polymeric and/or non-polymericstructures. It is also to be understood that the conjugate frameworksmay be linear, branched, hyperbranched and/or a combination of these.The following section describes some exemplary conjugate frameworks.

In various embodiments, a conjugate may be prepared from a frameworkthat includes a polymeric structure. For example, a polymer with pendantreactive groups (e.g., carboxyl or reactive ester, amine, hydroxyl,aldehyde, sulfhydryl, maleimidyl, alkynyl, azido, etc.) may be employed.It will be appreciated that different pendant groups may be mixed in asingle framework (e.g., by co-polymerizing appropriate monomers indesired ratios to produce a polymeric framework). As discussed below,these reactive groups may be used to attach affinity ligands and/ordrugs to the framework. Co-polymers, mixtures, and adducts of differentframeworks may also be used. Such combinations may be useful foroptimizing the mechanical and chemical properties of a material.

In various embodiments, frameworks having carboxyl (or reactive ester)pendant groups (—COOH bearing frameworks, or CBFs) may be used. Suchframeworks may naturally include carboxyl groups or may be modified toinclude them. Exemplary polymeric CBFs include but are not limited tocarboxylated polysaccharides (CPS) such as alginate (Ag),carboxymethylated-D-manno-D-glucan (CMMG, available from DaiichiPharmaceutical Co.), carboxymethyldextran (CMDex), carboxymethylchitin(CMCh, available from Katakura Chikkalin Co.), N-desulfated N-acetylatedheparin (DSH), and hyaluronic acid (HA). DSH and CMDex may besynthesized according to Sugahara, et al., Biol. Pharm. Bull., 24,535-543 (2001). In general, hydroxylated frameworks may be carboxylatedthrough reaction with chloroacetic acid under basic conditions. In thecase of a polymeric framework the degree of carboxyl substitution withrespect to monomer may vary between 1 and 100 mol %. Naturally occurringcarboxylated polymers include but are not limited to carboxylatedpoly(amino acids) (CPAA) such as poly-L-glutamate and poly-L-aspartate.The carboxylate content may be varied between 1 and 100% mol COOH/mol AAresidue by copolymerizing carboxylated amino acids (e.g., amino acidswith a carboxyl group in addition to the carboxyl group which becomespart of the polymer backbone) with non-carboxylated amino acids (e.g.,amino acids whose only carboxyl group becomes part of the polymerbackbone).

In various embodiments, frameworks having amine pendant groups (—NH₂bearing frameworks, or NBFs) may be used. Such frameworks may benaturally occurring or may be chemically modified to include a primaryamine. The latter include but are not limited to polymeric frameworks,e.g., amine pendant polysaccharides (NPS) such as deacetylated chitosan(Ch) (Sigma Aldrich, Milwaukee, Wis.) and diethylaminoethyl etherdextran (DEAEDex), MW 500,000 g/mol (Polysciences, Warrington, Pa.). Inthe case of such polymeric frameworks the degree of amine substitutionwith respect to monomer may vary between 1 and 100 mol %. Other suitableNBFs include, but are not limited to, polynucleotides where one or moreof the purine bases has been derivatized with an amine group at the 2′location. Naturally occurring aminated polymers include but are notlimited to poly(amino acids) such as poly-L-lysine (PLL) and itsenantiomer. The amine content may be varied between 1 and 100% molNH₂/mol amino acid residue by copolymerizing an aminated amino acid(e.g., an amino acid with an amine in addition to the amine group thateventually becomes part of the polymer backbone) with non-aminated aminoacids (e.g., an amino acid whose only amine is that which eventuallybecomes part of the polymer backbone).

In various embodiments, polymers having hydroxyl pendant groups (—OHbearing frameworks, or OBFs) may be used. Such frameworks may benaturally hydroxylated or may be chemically modified to include ahydroxyl group. In addition to dextran, naturally occurring polymericOBFs include but are not limited to polysaccharides such as yeast mannan(Mn), pullulan (Pl), amylose (Am), amylopectin (AmP), glycogen (Gl),cellulose (Cl), hyaluronate (Hy), chondroitin (ChD), and dextrin (Dx),all of which may be obtained commercially from Sigma Aldrich. Inaddition, poly(amino acids) such as poly(serine), poly(threonine),poly(tyrosine), and poly(4-hydroxyproline) may also be employed ashydroxylated polymers. The hydroxyl content of the poly(amino acids) maybe varied between 1 and 100% mol —OH/mol amino acid residue byco-polymerizing hydroxylated amino acids with non-hydroxylated aminoacids. Of course, carboxyl (or reactive ester), amino, and hydroxylpendant groups may be mixed in a single polymer by co-polymerizing theappropriate amino acids in desired ratios.

In various embodiments, frameworks having sulfhydryl pendant groups (—SHbearing frameworks, or SBFs) may be used. SBFs may be naturallysulfhydrylated or may be chemically modified using standard organicchemistry techniques to include a sulfhydryl group. In otherembodiments, frameworks having aldehyde, maleimidyl, alkynyl, azido,etc. pendant groups may be used.

In addition to the aforementioned classes of frameworks, some exemplarypolymers that may be used include poly(lactic acid) (PLA), poly(glycolicacid) (PGA), PLA-PGA co-polymers (PLGA), poly(anhydrides), poly(hydroxyacids), poly(ortho esters), poly(propylfumerates), poly(caprolactones),polyamides, polyacetals, biodegradable polycyanoacrylates andbiodegradable polyurethanes.

In various embodiments, conjugates of the following general formula (I)may be employed:

Various embodiments of the conjugates of formula (I) are described inmore detail in Example 9; however, in general it is to be understoodthat:

-   -   R^(x) is hydrogen or optionally substituted C₁₋₆ alkyl;    -   Z¹ is an optionally substituted bivalent C₁₋₁₀ hydrocarbon        chain, wherein 1, 2, 3, 4 or 5 methylene units of Z¹ are        optionally and independently replaced with one or more groups        selected from —S—, —O—, —NR^(a)—, —(C═NR^(a))—, —(C═O)—,        —(S═O)—, —S(═O)₂—, —(CR^(b)═CR^(b))—, —(N═N)—, an optionally        substituted arylene moiety or an optionally substituted        heteroarylene moiety, wherein R^(a) is hydrogen, optionally        substituted aliphatic, optionally substituted heteroaliphatic,        optionally substituted aryl, optionally substituted heteroaryl,        or a suitable amino protecting group; and R^(b) is hydrogen,        optionally substituted aliphatic, optionally substituted        heteroaliphatic, optionally substituted aryl, or optionally        substituted heteroaryl;    -   each occurrence of X¹ is independently —OR^(c) or —N(R^(d))₂,        wherein R^(c) is hydrogen, optionally substituted aliphatic,        optionally substituted heteroaliphatic, optionally substituted        aryl, optionally substituted heteroaryl, a suitable hydroxyl        protecting group, a cation group, or an affinity ligand, and        each R^(d) is, independently, hydrogen, optionally substituted        aliphatic, optionally substituted heteroaliphatic, optionally        substituted aryl, optionally substituted heteroaryl, a suitable        amino protecting group, or an affinity ligand, with the proviso        that at least two occurrences of X¹ include an affinity ligand;    -   Y¹ is hydrogen, halogen, optionally substituted aliphatic,        optionally substituted heteroaliphatic, optionally substituted        aryl, optionally substituted heteroaryl, —OR^(e) or —SR^(e)        wherein R^(e) is hydrogen, optionally substituted aliphatic,        optionally substituted heteroaliphatic, optionally substituted        aryl, or optionally substituted heteroaryl;    -   r is an integer between 5-25, inclusive;    -   W¹ is a drug; and    -   corresponds to a single or double covalent bond.

In various embodiments, conjugates of the following general formula (II)may be employed:

wherein:

-   -   each occurrence of

represents a potential branch within the conjugate;

-   -   each occurrence of

represents a potential repeat within a branch of the conjugate;

-   -   each occurrence of

is independently a covalent bond, a carbon atom, a heteroatom, or anoptionally substituted group selected from the group consisting of acyl,aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic;

-   -   each occurrence of T is independently a covalent bond or a        bivalent, straight or branched, saturated or unsaturated,        optionally substituted C₁₋₃₀ hydrocarbon chain wherein one or        more methylene units of T are optionally and independently        replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—,        —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a        heterocyclic group, an aryl group, or a heteroaryl group;    -   each occurrence of R is independently hydrogen, a suitable        protecting group, or an acyl moiety, arylalkyl moiety, aliphatic        moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic        moiety;    -   —B is -T-L^(B)-X;    -   each occurrence of X is independently an affinity ligand;    -   each occurrence of L^(B) is independently a covalent bond or a        group derived from the covalent conjugation of a T with an X;    -   -D is -T-L^(D)-W;    -   each occurrence of W is independently a drug;    -   each occurrence of L^(D) is independently a covalent bond or a        group derived from the covalent conjugation of a T with a W;    -   k is an integer from 2 to 11, inclusive, defining at least two        k-branches within the conjugate;    -   q is an integer from 1 to 4, inclusive;    -   k+q is an integer from 3 to 12, inclusive;    -   each occurrence of p is independently an integer from 1 to 5,        inclusive; and    -   each occurrence of n is independently an integer from 0 to 5,        inclusive; and    -   each occurrence of m is independently an integer from 1 to 5,        inclusive; and    -   each occurrence of v is independently an integer from 0 to 5,        inclusive, with the proviso that within each k-branch at least        one occurrence of n is ≧1 and at least one occurrence of v is        ≧1.

It is to be understood that general formula (II) (and other formulasherein) does not expressly list every hydrogen. For example, if thecentral

is a C₆ aryl group and k+q<6 it will be appreciated that the openposition(s) on the C₆ aryl ring include a hydrogen.In general, it will be appreciated that each occurrence of

represents a potential branching node and that the number of branches ateach node are determined by the values of k for the central

and n for non-central occurrences of

Since k≧2 the conjugate will always include at least two k-branches. Oneof ordinary skill will appreciate that because each occurrence of n maybe an integer from 0 to 5, the present disclosure contemplates bothbranched and hyperbranched (e.g., dendrimer-like) embodiments of theseconjugates. The proviso which requires that within each k-branch atleast one occurrence of n is ≧1 and at least one occurrence of v is ≧1ensures that every conjugate includes at least two separate k-brancheswith an occurrence of B (i.e., an affinity ligand).

In certain embodiments, each occurrence of

in a p-bracketed moiety is substituted by a number of n-bracketedmoieties corresponding to a value of n≧1. For example, when k=2 and p=2in both k-branches, the conjugate may be of the formula (IIa):

In other embodiments, only terminal occurrences of

in a p-bracketed moiety are substituted by a number of n-bracketedmoieties corresponding to a value of n≧1. For example, when k=2 and p=2in both k-branches (and n=0 for the first p-bracketed moiety in bothk-branches), the conjugate may be of the formula (IIb):

In certain embodiments, each occurrence of

in an m-bracketed moiety is substituted by a number of B moietiescorresponding to the value of v≧1. For example, when k=2, eachoccurrence of p=1, and each occurrence of m=2, the conjugate may be ofthe formula (IIc):

In other embodiments, only terminal occurrences of

in an m-bracketed moiety are substituted by a number of B moietiescorresponding to a value of v≧1. For example, when k=2, each occurrenceof p=1, and each occurrence of m=2 (and v=0 for the first m-bracketedmoiety in each n-branch), the conjugate may be of the formula (IId):

By way of further example, when q=1 and n=1 in both k-branches of theprevious formula, the conjugate may be of the formula (IIe):

Alternatively, when q=1 and n=2 in both k-branches of the previousformula, the conjugate may be of the formula (IIf):

In various embodiments, the present disclosure also provides conjugateswhich include affinity ligands and/or a drug which are non-covalentlybound the conjugate framework.

For example, in some embodiments, the present disclosure providesconjugates of any of the foregoing formulas, wherein:

-   -   each of

T, D, k, q, k+q, p, n, m and v is defined as described above and herein;

-   -   —B is -T-LRP^(B)-X;    -   each occurrence of X is independently an affinity ligand; and    -   each occurrence of LRP^(B) is independently a ligand-receptor        pair which forms a non-covalent bond between T and X with a        dissociation constant in human serum of less than 1 pmol/L.

In yet other embodiments, the present disclosure provides conjugates ofany of the foregoing formulas, wherein:

-   -   each of

T, B, k, q, k+q, p, n, m and v is defined as described above and herein;

-   -   -D is -T-LRP^(D)-W;    -   each occurrence of W is independently a drug; and    -   each occurrence of LRP^(D) is independently a ligand-receptor        pair which forms a non-covalent bond between T and W with a        dissociation constant in human serum of less than 1 pmol/L.

In other embodiments, the present disclosure provides conjugates of anyof the foregoing formulas wherein:

-   -   each of

T, k, q, k+q, p, n, m and v is defined as described above and herein;

-   -   —B is -T-LRP^(B)-X;    -   each occurrence of X is independently an affinity ligand;    -   each occurrence of LRP^(B) is independently a ligand-receptor        pair which forms a non-covalent bond between T and X with a        dissociation constant in human serum of less than 1 pmol/L.    -   -D is -T-LRP^(D)-W;    -   each occurrence of W is independently a drug; and    -   each occurrence of LRP^(D) is independently a ligand-receptor        pair which forms a non-covalent bond between T and W with a        dissociation constant in human serum of less than 1 pmol/L.

In various embodiments, a conjugate of the present disclosure may havethe general formula (III):

wherein

B, T, D, v, m, n, and p are as defined and described herein, k is aninteger from 1 to 11, inclusive, and j is an integer from 2 to 4,inclusive. Conjugates of formula (III) may have multiple sites ofconjugation of ligand to drug. It will be appreciated that, when q is 1,similar subgenera described to those described above (formulae (IIa) to(IIf)) can be contemplated by one skilled in the art for conjugates offormula (III) wherein j is 2, 3, or 4.

For purposes of exemplification and for the avoidance of confusion it isto be understood that an occurrence of:

in a conjugate of formula (III) (i.e., when j is 2) could be representedas:

(when the drug is covalently bound to the conjugate framework) or

(when the drug is non-covalently bound to the conjugate framework).

Description of Exemplary Groups

(node)

In certain embodiments, each occurrence of

is independently an optionally substituted group selected from the groupconsisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, andheterocyclic. In some embodiments, each occurrence of

is the same. In some embodiments, the central

is different from all other occurrences of

In certain embodiments, all occurrences of

are the same except for the central

In some embodiments,

is an optionally substituted aryl or heteroaryl group. In someembodiments,

is 6-membered aryl. In certain embodiments,

is phenyl. In certain embodiments,

is a heteroatom selected from N, O, or S. In some embodiments,

is nitrogen atom. In some embodiments,

is an oxygen atom. In some embodiments,

is sulfur atom. In some embodiments,

is a carbon atom.

T (Spacer)

In certain embodiments, each occurrence of T is independently abivalent, straight or branched, saturated or unsaturated, optionallysubstituted C₁₋₂₀ hydrocarbon chain wherein one or more methylene unitsof T are optionally and independently replaced by —O—, —S—, —N(R)—,—C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—,—N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an aryl group, or aheteroaryl group. In certain embodiments, one, two, three, four, or fivemethylene units of T are optionally and independently replaced. Incertain embodiments, T is constructed from a C₁₋₁₀, C₁₋₈, C₁₋₆, C₁₋₄,C₂₋₁₂, C₄₋₁₂, C₆₋₁₂, C₈₋₁₂, or C₁₀₋₁₂ hydrocarbon chain wherein one ormore methylene units of T are optionally and independently replaced by—O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—,—S(O)—, —S(O)₂—, —N(R)SO₂—, —SO₂N(R)—, a heterocyclic group, an arylgroup, or a heteroaryl group. In some embodiments, one or more methyleneunits of T is replaced by a heterocyclic group. In some embodiments, oneor more methylene units of T is replaced by a triazole moiety. Incertain embodiments, one or more methylene units of T is replaced by—C(O)—. In certain embodiments, one or more methylene units of T isreplaced by —C(O)N(R)—. In certain embodiments, one or more methyleneunits of T is replaced by —O—.

In some embodiments, T is

In some embodiments, T is

In some embodiments, T is

In some embodiments, T is

In some embodiments, T is

In some embodiments, T is

In certain embodiments, each occurrence of T is the same.

In certain embodiments, each occurrence of T (outside groups B and D) isa covalent bond and the conjugate is of the general formula (V) or (VI):

wherein

B, D, v, m, n, p, k, and j are as defined and described for formula (II)or (III), respectively.

In certain embodiments of general formulae (V) and (VI), each occurrenceof

except for the central

is a covalent bond, each occurrence of v=1, and the conjugate is of theformula (VII) or (VIII):

wherein

B, D, q, k, and j are as defined and described for formula (II) or(III), respectively.

In certain such embodiments for formula (VII), k=2 and q=1.

In other embodiments, k=3 and q=1.

In other embodiments, k=2 and q=2.

In certain such embodiments for formula (VIII), k=1 and j=2.

In other embodiments, k=2 and j=2.

In other embodiments, k=3 and j=2.

In other embodiments, k=1 and j=3.

In other embodiments, k=2 and j=3.

In other embodiments, k=3 and j=3.

In some embodiments, the present disclosure provides conjugates ofgeneral formula (VIIa):

wherein B and D are as defined and described herein.

For example, in some embodiments, the present disclosure providesconjugates of formula:

wherein W and X is as defined and described herein.

In some embodiments, the present disclosure provides conjugates ofgeneral formula (VIIb):

wherein B and D are as defined and described herein.

For example, in some embodiments, the present disclosure providesconjugates of formula:

wherein W and X are as defined and described herein.

In some embodiments, the present disclosure provides conjugates ofgeneral formula (VIIc):

wherein B and D are as defined and described herein.

For example, in some embodiments, the present disclosure providesconjugates of formula:

wherein W and X are as defined and described herein.

It will be appreciated that similar subgenera to those of formulae(VIIa), (VIIb), and (VIIc), and species thereof, can be contemplated byone skilled in the art for conjugates of formula (VIII) wherein j is 2,3, or 4. For example, when j is 2, in certain embodiments, the presentdisclosure provides conjugates of formula:

wherein B and D are as defined and described herein.

In certain embodiments, the present disclosure provides conjugates offormula:

wherein W, X, and j are as defined and described herein.

B (Ligand)

In various embodiments, —B is -T-L^(B)-X where X is a ligand; and L^(B)is a covalent bond or a group derived from the covalent conjugation ofan X with a T. Exemplary ligands were described above.

D (Drug)

In various embodiments, -D is -T-L^(D)-W where W is a drug and L^(D) isa covalent bond or a group derived from the covalent conjugation of a Wwith a T. Exemplary drugs were described above.

L^(B) and L^(D) (Covalent Conjugation)

One of ordinary skill will appreciate that a variety of conjugationchemistries may be used to covalently conjugate an X with a T and/or a Wwith a T (generally “components”). Such techniques are widely known inthe art, and exemplary techniques are discussed below. Components can bedirectly bonded (i.e., with no intervening chemical groups) orindirectly bonded through a spacer (e.g., a coupling agent or covalentchain that provides some physical separation between the conjugatedelement and the remainder of the conjugate framework). It is to beunderstood that components may be covalently bound to a conjugateframework through any number of chemical bonds, including but notlimited to amide, amine, ester, ether, thioether, isourea, imine, etc.bonds. In certain embodiments, L^(B) and/or L^(D) (generally “L” for thepurposes of this section) is a covalent bond. In some embodiments, L isan optionally substituted moiety derived from conjugating an optionallysubstituted carbonyl-reactive, thiol-reactive, amine-reactive, orhydroxyl-reactive moiety of T with a carboxyl, thiol, amine, or hydroxylgroup of X or W. In some embodiments, L is an optionally substitutedmoiety derived from conjugating an optionally substitutedcarboxyl-reactive, thiol-reactive, amine-reactive, or hydroxyl-reactivemoiety of X or W with a carboxyl, thiol, amine, or hydroxyl group of T.In some embodiments, L is

In some embodiments, L is a succinimide moiety.

In various embodiments, components may be covalently bound to aconjugate framework using “click chemistry” reactions as is known in theart. These include, for example, cycloaddition reactions, nucleophilicring-opening reactions, and additions to carbon-carbon multiple bonds(e.g., see Kolb and Sharpless, Drug Discovery Today 8:1128-1137, 2003and references cited therein as well as Dondoni, Chem. Asian J.2:700-708, 2007 and references cited therein). As discussed above, invarious embodiments, the components may be bound to a conjugateframework via natural or chemically added pendant groups. In general, itwill be appreciated that the first and second members of a pair ofreactive groups (e.g., a carboxyl group and an amine group which reactto produce an amide bond) can be present on either one of the componentand framework (i.e., the relative location of the two members isirrelevant as long as they react to produce a conjugate). Exemplarylinkages are discussed in more detail below. In various embodiments,carboxyl (or reactive ester) bearing components can be conjugated to —OHbearing frameworks (OBFs) using the procedure outlined by Kim et al.,Biomaterials 24:4843-4851 (2003). Briefly, the OBF is dissolved in DMSOalong with the carboxyl bearing component and reacted by means ofN′,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP)as catalysts under a dry atmosphere. Carboxyl bearing components can beconjugated to —NH₂ bearing frameworks (NBFs) using a carbodiimide (EDAC)coupling procedure. Using this procedure, the carboxyl bearing componentis functionalized by reaction with EDAC in a pH 5 buffer followed by theaddition of the NBF. In either of these cases (and in any of thefollowing cases), the resulting products may be purified by any numberof means available to those skilled in the art including, but notlimited to, size exclusion chromatography, reversed phasechromatography, silica gel chromatography, ion exchange chromatography,ultrafiltration, and selective precipitation.

In various embodiments, amine bearing components can be coupled to —COOHbearing frameworks (CBFs). CBFs using activated ester moieties (e.g.,see Hermanson in Bioconjugate Techniques, 2^(nd) edition, AcademicPress, 2008 and references cited therein). Briefly, a CBF with terminalactivated carboxylic acid esters such as —NHS, —SSC, —NPC, etc. isdissolved in an anhydrous organic solvent such as DMSO or DMF. Thedesired number of equivalents of amine bearing component are then addedand mixed for several hours at room temperature. Amine bearingcomponents can also be conjugated to CBFs to produce a stable amide bondas described by Baudys et al., Bioconj. Chem. 9:176-183, 1998. Thisreaction can be achieved by adding tributylamine (TBA) andisobutylchloroformate to a solution of the CBF and an amine bearingcomponent in dimethylsulfoxide (DMSO) under anhydrous conditions. Aminebearing components can alternatively be coupled to OBFs throughcyanalation using reagents including, but not limited to, cyanogenbromide (CNBr), N-cyanotriethylammonium tetrafluoroborate (CTEA),1-Cyano-4-(Dimethylamino)-pyridinium tetrafluorborate (CDAP), andp-nitrophenylcyanate (pNPC). CNBr reactions can be carried out at mildlybasic pH in aqueous solution. CDAP reactions are carried out in amixture of DMSO and water at mildly basic pH using triethylamine (TEA)as a catalyst. In certain embodiments, amine bearing components can beconjugated to NBFs, e.g., through glutaraldehyde coupling in aqueousbuffered solutions containing pyridine followed by quenching withglycine. In certain embodiments, amine bearing components can beconjugated to aldehyde bearing frameworks using a Schiff Base couplingprocedure followed by reduction (e.g., see Hermanson in BioconjugateTechniques, 2^(nd) edition, Academic Press, 2008 and references citedtherein as well as Mei et al. in Pharm. Res. 16:1680-1686, 1999 andreferences cited therein). Briefly, a framework with terminal activatedaldehydes (e.g., acetaldehyde, propionaldehyde, butyraldehyde, etc.) isdissolved in an aqueous buffer with the pH at or below neutral toprevent unwanted aldehyde hydrolysis. The desired number of equivalentsof an amine bearing component are then added and mixed at roomtemperature followed by addition of an excess of suitable reducing agent(e.g., sodium borohydride, sodium cyanobrohydride, sodiumtriacetoxyborohydride pyridine borane, triethylamine borane, etc.).

In various embodiments, hydroxyl bearing components can be conjugated toOBFs according to the divinylsulfone (DVS) procedure. Using thisprocedure, the OBF is added to a pH 11.4 bicarbonate buffer andactivated with DVS followed by addition of a hydroxyl bearing componentafter which glycine is added to neutralize and quench the reaction.Hydroxyl bearing components may also be coupled to OBFs using activatedester moieties as described above to produce ester bonds.

In various embodiments, sulfhydryl bearing components can be coupled tomaleimide bearing frameworks (MBFs) using a relatively mild procedure toproduce thioether bonds (e.g., see Hermanson in Bioconjugate Techniques,2^(nd) edition, Academic Press, 2008 and references cited therein).Because the maleimide group is much less susceptible to hydrolysis thanactivated esters, the reaction can be carried out under aqueousconditions. Briefly, an MBF is dissolved in a buffered aqueous solutionat pH 6.5-7.5 followed by the desired number of equivalents ofsulfhydryl bearing component. After mixing at room temperature forseveral hours, the thioether coupled conjugate may be purified.Sulfhydryl bearing components can also be conjugated to NBFs accordingto a method described by Thoma et al., J. Am. Chem. Soc. 121:5919-5929,1999. This reaction involves suspending the NBF in anhydrousdimethylformamide (DMF) followed by the addition of 2,6-lutidine andacid anhydride and subsequent purification of the reactive intermediate.A sulfhydryl bearing component is then added to a solution of theintermediate in DMF with triethylamine.

In various embodiments, azide bearing components can be coupled to analkyne bearing framework (ABF) using the copper(I)-catalyzed modernversion of the Huisgen-type azide-alkyne cycloaddition to give a1,4-di-substituted 1,2,3-triazole (e.g., see Dondoni, Chem. Asian J.2:700-708, 2007 and references cited therein as well as Dedola et al.,Org. Biomol. Chem. 5: 1006-1017, 2007). This reaction, commonly referredto as a “click” reaction, may be carried out for example in neat THFusing N,N-diisopropylethylamine and Cu(PPh₃)₃Br as the catalyst system(e.g., see Wu et al., Chem. Commun. 5775-5777, 2005). The reaction mayalso be carried out in a 3:1 (THF:water) mixture using sodium ascorbateand CuSO₄.5H₂O as the catalyst system (e.g., see Wu et al., supra). Ineither case, the azide bearing component is added to the ABF at thedesired number of equivalents followed by mixing for 12-48 hours at roomtemperature. Alternatively, alkyne bearing components may be conjugatedto an azide bearing framework using exactly the same conditionsdescribed above.

Certain components may naturally possess more than one of the samechemically reactive moiety. In some examples, it is possible to choosethe chemical reaction type and conditions to selectively react thecomponent at only one of those sites. For example, in the case whereinsulin is conjugated through reactive amines, in certain embodiments,the N-terminal α-Phe-B1 is a preferred site of attachment over theN-terminal α-Gly-A1 and ε-Lys-B29 to preserve insulin bioactivity (e.g.,see Mei et al., Pharm. Res. 16: 1680-1686, 1999 and references citedtherein as well as Tsai et al., J. Pharm. Sci. 86: 1264-1268, 1997). Inan exemplary reaction between insulin with hexadecenal (analdehyde-terminated molecule), researchers found that mixing the twocomponents overnight in a 1.5M pH 6.8 sodium salicylate aqueous solutioncontaining 54% isopropanol at a ratio of 1:6 (insulin:aldehyde mol/mol)in the presence of sodium cyanoborohydride resulted in over 80%conversion to the single-substituted Phe-B1 secondary amine-conjugatedproduct (Mei et al., Pharm. Res. 16:1680-1686, 1999). Their studiesshowed that the choice of solvent, pH, and insulin:aldehyde ratio allaffected the selectivity and yield of the reaction. In most cases,however, achieving selectivity through choice of chemical reactionconditions is difficult. Therefore, in certain embodiments it may beadvantageous to selectively protect the component (e.g., insulin) at allsites other than the one desired for reaction followed by a deprotectionstep after the material has been reacted and purified. For example,there are numerous examples of selective protection of insulin aminegroups available in the literature including those that may bedeprotected under acidic (BOC), slightly acidic (citraconic anhydride),and basic (MSC) conditions (e.g., see Tsai et al., J. Pharm. Sci. 86:1264-1268, 1997; Dixon et al., Biochem. J. 109: 312-314, 1968; andSchuettler et al., D. Brandenburg Hoppe Seyler's Z. Physiol. Chem. 360:1721, 1979). In one example, the Gly-A1 and Lys-B29 amines may beselectively protected with tert-butoxycarbonyl (BOC) groups which arethen removed after conjugation by incubation for one hour at 4 C in a90% trifluoroacetic acid (TFA)/10% anisole solution. In one embodiment,a dry powder of insulin is dissolved in anhydrous DMSO followed by anexcess of triethylamine. To this solution, approximately two equivalentsof di-tert-butyl dicarbonate solution in THF is added slowly and thesolution allowed to mix for 30-60 minutes. After reaction, the crudesolution is poured in an excess of acetone followed by dropwise additionof dilute HCl to precipitate the reacted insulin. The precipitatedmaterial is centrifuged, washed with acetone and dried completely undervacuum. The desired di-BOC protected product may be separated fromunreacted insulin, undesired di-BOC isomers, and mono-BOC and tri-BOCbyproducts using preparative reverse phase HPLC or ion exchangechromatography (e.g., see Tsai et al., J. Pharm. Sci. 86: 1264-1268,1997). In the case of reverse phase HPLC, a solution of the crudeproduct in 70% water/30% acetonitrile containing 0.1% TFA is loaded ontoa C8 column and eluted with an increasing acetonitrile gradient. Thedesired di-BOC peak is collected, rotovapped to remove acetonitrile, andlyophilized to obtain the pure product.

LRP^(B) and LRP^(D) (Non-Covalent Conjugation)

One of ordinary skill will appreciate that a variety of conjugationchemistries may be used to non-covalently conjugate an X with a T and/orW with a T (generally “components”). Such techniques are widely known inthe art, and exemplary techniques are discussed below. In certainembodiments, the dissociation constant (K_(d)) of the non-covalentlinkage in human serum is less than 1 pmol/L. For example, a componentmay be non-covalently bound to a conjugate framework via a non-covalentligand-receptor pair as is well known in the art (e.g., withoutlimitation a biotin-avidin based pair). In such an embodiment, onemember of the ligand receptor-pair is covalently bound to the componentwhile the other member of the pair is covalently bound to the conjugateframework. When the component and conjugate framework are combined, thestrong non-covalent interaction between the ligand and its receptorcauses the component to become non-covalently bound to the conjugateframework. Typical ligand/receptor pairs include protein/co-factor andenzyme/substrate pairs. Besides the commonly used biotin/avidin pair,these include without limitation, biotin/streptavidin,digoxigenin/anti-digoxigenin, FK506/FK506-binding protein (FKBP),rapamycin/FKBP, cyclophilin/cyclosporin and glutathione/glutathionetransferase pairs. Other suitable ligand/receptor pairs would berecognized by those skilled in the art, e.g., monoclonal antibodiespaired with a epitope tag such as, without limitation,glutathione-S-transferase (GST), c-myc, FLAG® and further thosedescribed in Kessler pp. 105-152 of Advances in Mutagenesis” Ed. byKessler, Springer-Verlag, 1990; “Affinity Chromatography: Methods andProtocols (Methods in Molecular Biology)” Ed. by Pascal Baillon, HumanaPress, 2000; and “Immobilized Affinity Ligand Techniques” by Hermansonet al., Academic Press, 1992.

k and q

For conjugates of general formula (II), k is an integer from 2 to 11,inclusive, defining at least two k-branches within the conjugate. Incertain embodiments, k=2 or 3. q is an integer from 1 to 4, inclusive,and defines the number of D groups which are bound to the central

group. In certain embodiments, q=1. In some embodiments, q=2. k+q is aninteger from 3 to 6, inclusive. In certain embodiments, k+q=3 or 4.

For conjugates of general formula (III), when j is 2, 3, or 4, k is aninteger from 1 to 11, inclusive. In certain embodiments, k is 1, 2, or3. q is an integer from 1 to 4, inclusive, and defines the number of Dgroups which are bound to the central

group. In certain embodiments, q=1. In some embodiments, q=2. k+q is aninteger from 3 to 6, inclusive. In certain embodiments, k+q=3 or 4.p and m

Each occurrence of p is independently an integer from 1 to 5, inclusive.In certain embodiments, each occurrence of p is the same. In certainembodiments, p=1, 2 or 3. In certain embodiments, p=1.

Each occurrence of m is independently an integer from 1 to 5, inclusive.In certain embodiments, each occurrence of m is the same. In certainembodiments, m=1, 2 or 3. In certain embodiments, m=1.

n and v

Each occurrence of n is independently an integer from 0 to 5, inclusive,with the proviso that within each k-branch at least one occurrence of nis ≧1. Branches within a given k-branch are referred to herein asn-branches.

In certain embodiments, each occurrence of

in a p-bracketed moiety is substituted by a number of n-bracketedmoieties corresponding to a value of n≧1, e.g., see formula (IIa) above.In some such embodiments, each occurrence of n in the conjugate is thesame. In some of these embodiments, n=1 or 2.

In other embodiments, only terminal occurrences of

in a p-bracketed moiety are substituted by a number of n-bracketedmoieties corresponding to a value of n≧1, e.g., see formula (IIb) above.In certain embodiments, each k-branch includes just one occurrence ofn≧1 (i.e., all other occurrences of n=0). In some such embodiments, eachoccurrence of n in the conjugate is the same. In some of theseembodiments, n=1 or 2.

Each occurrence of v is independently an integer from 0 to 5, inclusive,with the proviso that within each k-branch at least one occurrence of vis ≧1.

In certain embodiments, each occurrence of

in an m-bracketed moiety is substituted by a number of B moietiescorresponding to the value of v≧1, e.g., see formula (IIc) above. Insome such embodiments, each occurrence of v in the conjugate is thesame. In some of these embodiments, v=1 or 2.

In other embodiments, only terminal occurrences of

in an m-bracketed moiety are substituted by a number of B moietiescorresponding to a value of v≧1, e.g., see formula (IId) above. Incertain embodiments, each k-branch includes just one occurrence of v≧1(i.e., all other occurrences of v=0). In some such embodiments, eachoccurrence of v in the conjugate is the same. In some of theseembodiments, v=1 or 2. In certain embodiments, each n-branch includes atleast one occurrence of v≧1. In certain embodiment, each n-branchincludes just one occurrence of v≧1 (i.e., all other occurrences ofv=0). In some such embodiments, each occurrence of v in the conjugate isthe same. In some of these embodiments, v=1 or 2.j

j of formula (II) is an integer from 1 to 4, inclusive, and defines thenumber of conjugations to the D group. In certain embodiments, j=1. Incertain embodiments, j=2. In some embodiments, j=3. In otherembodiments, j=4.

Drug Loading

In general, the amount of drug that is loaded onto a conjugate willdepend on the molecular weight of the drug and can be controlled byadjusting the molecular weight of the conjugate framework and/or thelevel of chemical activation (i.e., when pendant groups are added to theframework). In various embodiments, the drug loading level may be in therange of 5 to 99% w/w of drug to conjugate (i.e., including drug). Invarious embodiments, loading levels within the narrower range of 50 to99% may be used, e.g., in the range of 80 to 99%.

Other

In various embodiments, a biodegradable framework may be used. Invarious embodiments, a non-biodegradable framework may be used, e.g.,when biodegradability is not relevant to the application and/or when theresulting framework or conjugate is sufficiently well excreted thatbiodegradability is not necessary. In various embodiments, the conjugateframework (or spacer when present, e.g., between a drug and framework)is susceptible to digestion by an enzyme. In various embodiments, theenzyme is present at the site of administration. One skilled in the artwill recognize that a number of enzymes are present in patients thatcould cleave a conjugate framework. Without limitation, these includesaccharidases, peptidases, and nucleases. Exemplary saccharidasesinclude, but are not limited to, maltase, sucrase, amylase, glucosidase,glucoamylase, and dextranase. Exemplary peptidases include, but are notlimited to, dipeptidyl peptidase-IV, prolyl endopeptidase, prolidase,leucine aminopeptidase, and glicyl glycine dipeptidase. Exemplarynucleases include, but are not limited to, deoxyribonuclease I,ribonuclease A, ribonucelase T1, and nuclease S1.

One skilled in the art will also recognize that, depending on the choiceof enzyme, there are a number of conjugate frameworks that aresusceptible to enzymatic cleavage. For example, in cases wheresaccharidase degradation is desired, frameworks which includepolysaccharides can be used (e.g., without limitation, a conjugate thatincludes a polysaccharide comprising repeating chains of 1,4-linkedalpha-D-glucose residues will be degraded by alpha-amylases). Withoutlimitation, suitable polysaccharides include glycogen and partiallydigested glycogen derived from any number of sources, including but notlimited to, sweet corn, oyster, liver (human, bovine, rabbit, rat,horse), muscle (rabbit leg, rabbit abdominal, fish, rat), rabbit hair,slipper limpet, baker's yeast, and fungus. Other polysaccharide polymersand spacers that one could use include carboxylated polysaccharides,—NH₂ pendant polysaccharides, hydroxylated polysaccharides, alginate,collagen-glycosaminoglycan, collagen, mannan, amylose, amylopectin,cellulose, hyaluronate, chondroitin, dextrin, chitosan, etc. In caseswhere peptidase cleavage is desired, polypeptides that contain aminoacid sequences recognized by the cleaving enzyme can be used (e.g.,without limitation, a conjugate that includes a [-Glycine-Proline-]sequence will be degraded by prolidase). In certain embodiments onecould use co-polymers of aminated and non-aminated amino acids,co-polymers of hydroxylated and non-hydroxylated amino acids,co-polymers of carboxylated and non-carboxylated amino acids,co-polymers of the above or adducts of the above. In cases wherenuclease degradation is desired, polynucleotides can be used (e.g.,without limitation, a conjugate that includes a polynucleotidecontaining an oligomer of sequential adenosine residues will be degradedby ribonuclease A).

In various embodiments, the pharmacokinetic and/or pharmacodynamicbehavior of a conjugate (i.e., conjugated drug and/or drug which hasbeen released from a conjugate by chemical or enzymatic degradation) maybe substantially the same as the corresponding unconjugated drug (e.g.,when both are administered subcutaneously). For example, from apharmacokinetic (PK) perspective, the serum concentration curve may besubstantially the same as when an equivalent amount of unconjugated drugis administered. Additionally or alternatively, the serum T_(max), serumC_(max), mean serum residence time (MRT), mean serum absorption time(MAT) and/or serum half-life may be substantially the same as when theunconjugated drug is administered. From a pharmacodynamic (PD)perspective, the conjugate may act on substances within the body insubstantially the same way as the unconjugated drug. For example, in thecase of an insulin conjugate, the conjugate may affect blood glucoselevels in substantially the same way as unconjugated insulin. In thiscase, substantially similar pharmacodynamic behavior can be observed bycomparing the time to reach minimum blood glucose concentration(T_(nadir)), the duration over which the blood glucose level remainsbelow a certain percentage of the initial value (e.g., 70% of initialvalue or T_(70% BGL)), etc. It will be appreciated that these PK and PDcharacteristics can be determined according to any of a variety ofpublished pharmacokinetic and pharmacodynamic methods (e.g., see Baudyset al., Bioconjugate Chem. 9:176-183, 1998 for methods suitable forsubcutaneous delivery).

In one embodiment, a conjugate (i.e., in isolated form withoutcross-linking agents) produces pharmacokinetic (PK) parameters such astime to reach maximum serum drug concentration (T_(max)), mean drugresidence time (MRT), serum half-life, and mean drug absorption time(MAT) that are within 40% of those values determined for theunconjugated drug. In various embodiments, a conjugate produces PKparameters that are within 35%, 30%, 25%, 20%, 15% or even 10% of thoseproduced by the unconjugated drug. In some embodiments, a conjugateproduces PK parameters that are within 20% of those produce by theunconjugated drug. For example, in embodiments involving an insulinconjugate for subcutaneous delivery the conjugate may produce an insulinT_(max) between 15-30 minutes, a mean insulin residence time (MRT) ofless than 50 minutes, or a mean insulin absorption time (MAT) of lessthan 40 minutes, all of which are within 20% of those values determinedfrom the human recombinant insulin treatment group. In certainembodiments, the conjugate may produce an insulin T_(max) between 20-25minutes, a mean insulin residence time (MRT) of less than 45 minutes,and a mean insulin absorption time (MAT) of less than 35 minutes. Incertain embodiment, the conjugate may produce a serum half-life of lessthan 120 minutes, e.g., less than 100 minutes.

In one embodiment, an inventive conjugate produces pharmacodynamic (PD)parameters such as time to reach minimum/maximum blood concentration ofa substance (T_(nadir)/T_(max)) or duration over which the blood levelof the substance remains below/above 70%/130% of the initial value(T_(70% BL)/T_(130% AL)). For example, in embodiments involving aninsulin conjugate for subcutaneous delivery the conjugate may produce aglucose T_(nadir) between 45-60 minutes and a glucose T_(70% BGL) ofless than 180 minutes, both of which are within 20% of those determinedfrom the human recombinant insulin treatment group. In certainembodiments the conjugate may produce a glucose T_(nadir) between 50-55minutes and a glucose T_(70% BGL) of less than 160 minutes. In variousembodiments, a conjugate produces PD parameters that are within 40%,35%, 30%, 25%, 20%, 15% or even 10% of those produced by theunconjugated drug. In some embodiments, a conjugate produces PDparameters that are within 20% of those produce by the unconjugateddrug.

Intermediates for Preparing Conjugates

In one aspect, the invention provides reagents for preparing conjugatesof the present disclosure. Thus, in various embodiments, a compound ofgeneral formula (II) is provided wherein:

-   -   each of

T, D, k, q, k+q, p, n, m and v is defined as described above and herein;

-   -   B is -T-L^(B′); and    -   each occurrence of L^(B′) is independently hydrogen, an        alkyne-containing moiety, an azide-containing moiety, or an        optionally substituted carbonyl-reactive, thiol-reactive,        amine-reactive, or hydroxyl-reactive moiety.

In other embodiments, a compound of general formula (II) is providedwherein:

-   -   each of

T, B, k, q, k+q, p, n, m and v is defined as described above and herein;

-   -   D is -T-L^(D′); and    -   each occurrence of L^(D′) is independently hydrogen, an        alkyne-containing moiety, an azide-containing moiety, or an        optionally substituted carbonyl-reactive, thiol-reactive,        amine-reactive, or hydroxyl-reactive moiety.

Methods for Preparing Conjugates

We have exemplified methods for preparing the aforementioned conjugatesusing insulin as an exemplary drug and aminoethylglucose (AEG),aminoethylmannose (AEM), aminoethylbimannose (AEBM), and/oraminoethyltrimannose (AETM) as exemplary affinity ligands. Withoutlimitation, conjugates with two affinity ligands and one drug moleculeand with short distances between all framework components may beprepared using tris(hydroxymethyl)aminomethane (Tris), Tris-succinimidylaminotriacetate (TSAT), tris-Succinimidyl-1,3,5-benzenetricarboxylate(TSB), and Benzene-1,3,5-tricarboxy-(N-4-butyric-NHS-ester)amide(TSB-C4) as conjugate frameworks. If more space between frameworkcomponents is desired then Succinimidyl (6-aminocaproyl)aminotriacetate(TSAT-C6), Succinimidyl (6-amino(PEO-6))aminotriacetate (TSAT-PEO-6),Benzene-1,3,5-tricarboxy-(N-6-aminocaproic-NHS ester)amide (TSB-C6), andBenzene-1,3,5-tricarboxy-(N-10-aminodecanoic-NHS ester)amide (TSB-C10)may be used. The TSAT-C6 spacer arm chemistry imparts more hydrophobiccharacter to the conjugate as compared to TSAT-PEO-6. For example, forpurposes of illustration, in one embodiment, both the affinity ligand(e.g., AEG, AEM, AEMB and AETM) and insulin may be reacted to a TSAT-C6framework through the terminal activated esters to produceinsulin-TSAT-C6-AEG-2, insulin-TSAT-C6-AEM-2, insulin-TSAT-C6-AEMB-2,and insulin-TSAT-C6-AETM-2 conjugates. The various affinity ligands aresynthesized ahead of time as discussed in the Examples. In addition, theA1 and B29 amino groups of insulin are BOC-protected as described in theExamples so that each insulin can only react at the Phe-B1 α-aminogroup. Approximately one equivalent of BOC-insulin as a 40-50 mg/mlsolution in DMSO is added at room temperature to a 50 mg/ml solution ofTSAT-C6 in DMSO containing excess triethylamine and allowed to react forapproximately one hour. Next, an excess of AEG, AEM, AEBM, and/or AETM(2-10 equivalents) as a 100 mg/ml solution in DMSO is added and allowedto react for an additional 2 hours. After reaction, the DMSO solution issuperdiluted by 10× into a pH 5 saline buffer after which the pH isadjusted to 8.0 and the solution passed through a Biogel P2 column toremove low molecular reactants and salts. The material eluting in thevoid fraction is concentrated using a 3K ultrafiltration apparatus afterwhich it is injected on a prep scale reverse phase HPLC column (C8,acetonitrile/water mobile phase containing 0.1% TFA) to purify thedesired product from unreacted BOC2-insulin. The desired elution peak iscollected pooled and rotovapped to remove acetonitrile followed bylyophilization to obtain a dry powder. Finally, the BOC protectinggroups are removed by dissolving the lyophilized powder in 90% TFA/10%anisole for one hour at 4 C followed by 10× superdilution in HEPES pH8.2 buffer containing 0.150M NaCl. The pH is adjusted to between 7.0 and8.0 using NaOH solution after which the material is passed through aBiogel P2 column to remove anisole, BOC, and any other contaminatingsalts. The deprotected, purified aqueous conjugate solution is thenconcentrated to the desired level and stored at 4 C until needed.

It will be appreciated that this exemplary procedure may be used toproduce other conjugates with different affinity ligands and drugs,different conjugation chemistries, different separations betweenframework components, and/or different valencies by substituting theTSAT-C6 framework with a different framework as described below.

For example, if yet more distance is required between frameworkcomponents and/or a preserved charge is required at the site ofconjugation, then an appropriately-sized amine-bearing diethyl acetal(e.g., aminopropionaldehyde diethyl acetal (APDA) or aminobutyraldehydediethyl acetal (ABDA)) may be conjugated to one of the reactive groupson the frameworks listed here followed by complete reaction of theremaining reactive groups with the affinity ligand of interest (e.g.AEM, AEBM, or AETM). A reactive aldehyde group can then be revealed fromthe diethyl acetal under acidic conditions followed by a reductiveamination with insulin to complete the drug conjugation step thenABDA-TSAT, ABDA-LCTSAT, etc. may be employed. In yet another example,tetrakis-(N-succinimidyl carboxypropyl)pentaerythritol (TSPE), may beused to attach three affinity ligands and one drug molecule forincreased multivalency. It will also be appreciated by those skilled inthe art that any of the above teachings may be used to producehyperbranched (e.g., dendrimer-like) conjugates with even higher ordervalencies. For example, Röckendorf and Lindhorst provide a comprehensivereview of current approaches for producing hyperbranched structures inTopics in Current Chemistry. 217: 202-238, 2001. Furthermore, ligandsalready containing a predetermined degree of multivalency may again bereacted according to the procedures described above to produce evenhigher orders of ligand multiplicity. For example, a divalent AEM-2,AEBM-2, or AETM-2 molecule containing a terminal reactive amine may beprepared by conjugating two of each affinity ligand to a suitableframework to which a reactive amine is also conjugated. A trivalentAEM-3, AEBM-3, or AETM-3 molecule containing a terminal reactive aminemay be prepared by conjugating three of each affinity ligand to asuitable framework to which a reactive amine is also conjugated. TheNH₂-divalent sugars may be reacted with the same frameworks describedabove to produce drug conjugates with 4 and 6 ligands per drug molecule.The NH₂-trivalent sugars may be reacted with the same frameworksdescribed above to produce drug conjugates with 6 and 9 ligands per drugmolecule.

In all cases, it should be recognized that a mixture of differentligands may be conjugated to the same drug via a multivalent frameworkby adjusting the framework chemistry, valency, and the ligand:frameworkstoichiometry. For example, Insulin-AEM-1-AEBM-1, Insulin-AEBM-1-AETM-1,Insulin AEM-2-AETM-2, and Insulin AEM-1-AETM-2 may all be synthesizedaccording to this mixed ligand method.

Finally, in some cases, it may be desirable to conjugate the affinityligand to the framework through a different means than the drug. Forexample, a divalent maleimide/monovalent activate ester functionalizedframework (e.g., succinimidyl-3,5-dimaleimidophenyl benzoate (SDMB)) maybe used to conjugate two sulfhydryl functionalized affinity ligands andone amine-functionalized drug in separate steps. For example, insulin oranother amine-containing drug may be conjugated to the activated esterportion of the framework using methods described herein. In a separatestep, the aminoethyl sugar (AEM, AEBM, AETM) may be converted to aterminal sulfhydryl-bearing ligand by reaction with 4-iminothiolane.Finally, the framework-di-maleimide-insulin conjugate may be mixed withan excess of sulfhydryl-functionalized sugar to produce the resultingdivalent-sugar-insulin conjugate.

Multivalent Cross-Linking Agents

The conjugates of the present disclosure are combined with multivalentcross-linking agents to form cross-linked materials. The followingsections describe exemplary cross-linking agents that can be used.

As discussed in more detail below and as illustrated in FIG. 4, thecross-linked material 10 is capable of controllably releasing theconjugates 20 in response to an exogenous target molecule. The materialsare prepared by combining the conjugates 20 with multivalentcross-linking agents 30 that non-covalently bind the affinity ligands 40of the conjugates 20 and thereby cross-link the conjugates 20 to formthe cross-linked material 10. The non-covalent bonds between themultivalent cross-linking agents 30 and the affinity ligands 40 arecompetitively dissociated in the presence of excess amounts of theexogenous target molecule.

1. Polypeptide Cross-Linking Agents

In various embodiments, the multivalent cross-linking agents may includea polypeptide. As discussed in more detail below, suitable multivalentpolypeptides exist in nature (e.g., various lectins) but can also beconstructed by linking multiple monovalent binding proteins, e.g.,monovalent lectins, peptide aptamers, antibodies, cell membranereceptors, etc. Still other multivalent polypeptides may be constructedby chemically linking binding fragments of these proteins.

A variety of mono- and multivalent ligand-binding proteins are availablecommercially (e.g., from Sigma-Aldrich), including a number of lectins,folate-binding protein, thyroxine-binding globulin, lactoferrin, etc.DeWolf and Best provide a review of ligand-binding proteins includingbiotin-binding proteins, lipid-binding proteins/transporters ofhydrophobic molecules, bacterial periplasmic binding proteins, lectins,serum albumins, immunoglobulins, inactivated enzymes, odorant-bindingproteins, immunosuppressant-binding proteins, and phosphate- andsulfate-binding proteins (see De Wolfe and Best, Pharm. Rev. 52:207-236, 2000 and references cited therein). The cell membrane receptorsfor a variety of hormones have also been described in the art. Incertain embodiments, mono- or multivalent binding proteins can besynthesized by rational computational design followed by site directedmutagenesis of existing ligand-binding proteins as described in Loogeret al., Nature 423:185-190, 2003. Exemplary protein fragments includetruncated MBP (Eda et al., Biosci. Biotechnol. Biochem., 62:1326-1331,1998), truncated conglutinin (Eda et al., Biochem. J. 316:43, 1996),truncated SP-D (Eda et al., Biochem. J. 323:393, 1997), and theglucose/galactose binding protein of E. Coli (Salins et al., AnalyticalBiochemistry 294:19-26, 2001).

a. Lectins

In certain embodiments, mono- or multivalent lectins may be included ina multivalent cross-linking agent. As discussed in more detail below, incertain embodiments, it may be advantageous to chemically modify thelectins. Lectins are particularly suitable for use in materials whichare designed to respond to a saccharide (e.g., α-methyl-mannose).Lectins have been isolated from a variety of natural sources includingseeds, roots, bark, fungi, bacteria, seaweed, sponges, mollusks, fisheggs, body fluids of invertebrates and lower vertebrates, and mammaliancell membranes (e.g., see The Lectins: Properties, Functions, andApplications in Biology and Medicine, Edited by Liener et al., AcademicPress, 1986). A number of lectins have also been produced recombinantly(e.g., see Streicher and Sharon, Methods Enzymol. 363:47-77, 2003 andU.S. Patent Publication No. 20060247154). As noted above, lectins bindsaccharides and polysaccharides with a high degree of specificity. Forexample, some lectins will bind only to mannose or glucose residues,while others only recognize galactose residues. Some lectins requirethat the particular residue be in a terminal position, while others bindto residues within a polysaccharide chain. Some lectins require specificanomeric structures and yet others recognize specific sugar sequences.The structures and properties of lectins have been extensively describedin the literature. For recent reviews see Lectins, Edited by Sharon andLis, Kluwer Academic Publishers, 2003; Handbook of Animal Lectins:Properties and Biomedical Applications, Edited by Kilpatrick, Wiley,2000; and Handbook of Plant Lectins: Properties and BiomedicalApplications, Edited by Van Damme et al., Wiley, 1998. Exemplary lectinsinclude calnexin, calreticulin, CD22, CD33, galectin (galactose-bindinglectin), myelin-associated glycoprotein, N-acetylglucosamine receptor,selectin, sialoadhesin, aggrecan, asialoglycoprotein receptor, CD94,collectin (mannose-binding lectin), mannose receptor, versican, abrin,ricin, concanavalin A, phytohaemagglutinin, and pokeweed mitogen. Invarious embodiments, human analogs of plant lectins may be used. Theseinclude, without limitation, human mannan binding protein (MBP, alsocalled mannan binding lectin, Sheriff et al., Structural Biology,1:789-794 (1994); Dumestre-Perard et al., Molecular Immunology,39:465-473 (2002)), human pulmonary surfactant protein A (SP-A, Allen,et al., Infection and Immunity, 67:4563-4569 (1999)), human pulmonarysurfactant protein D (SP-D, Persson et al., The Journal of BiologicalChemistry, 265:5755-5760 (1990)), CL-43 (a human serum protein), andconglutinin.

b. Peptide Aptamers

In certain embodiments monovalent peptide aptamers may be included in amultivalent cross-linking agent. As is well known in the art, peptideaptamers consist of a variable ligand-binding peptide loop fused withina protein scaffold (e.g., see Hoppe-Seyler and Butz, J. Mol. Med.78:426-430, 2000 and Crawford et al., Briefings in Functional Genomicsand Proteomics 2:72-79, 2003). The variable loop typically includesbetween about 10 and 20 amino acids. A variety of scaffold proteins maybe used. In general, the site of insertion is chosen such that thepeptide loop disrupts a region of the scaffold that would otherwisemediate some wild-type function, e.g., the bacterial proteinthioredoxin-A in which the variable loop is inserted within the reducingactive site (a -Cys-Gly-Pro-Cys-loop in the wild-type protein). Peptideaptamers with suitable affinity for the target molecule can be preparedand selected using any known method. For example, yeast two-hybridlibraries, yeast expression libraries, bacterial expression librariesand/or retroviral libraries for expression in mammalian cells may beused.

In various embodiments, peptide aptamers may be selected by affinitychromatography. According to such embodiments, peptide aptamers in alibrary are exposed to the target molecule and those that do not bindthe target are removed. The bound peptide aptamers are then eluted andcloned for subsequent rounds of selection. A new library is thengenerated from one or more of these peptide aptamers (e.g., the peptideaptamer with the highest affinity for the target molecule in the firstround of selection) and the stringency of the elution conditions isincreased or modified to identify peptide aptamers with the desiredbinding affinity and/or specificity. In various embodiments, theselection process may involve steps in which the stringency of theelution conditions are gradually increased in order to select peptideaptamers with high affinity for the target molecule. In variousembodiments, the selection process may involve steps in which theelution conditions are modified (e.g., by using a different affinitycolumn) in order to select peptide aptamers with desired specificity forthe target molecule. In various embodiments the selection process maygenerate a collection of sublibraries (or “pools”) each of whichcomprises peptide aptamers with similar affinities and/or specificitiesfor the target molecule. In various embodiments the selection processmay generate a single peptide aptamer sequence (or “monoclonal”). Itwill be appreciated that any of these peptide aptamer sequences may becloned for future recombinant expression.

c. Generating Multivalent Cross-Linking Agents

Multivalent cross-linking agents can be generated by covalently ornon-covalently linking two or more monovalent binding proteins into asingle construct. Typically, two or more proteins (which may have thesame or different sequences) may be linked directly to one another(e.g., via a coupling agent) or indirectly through a framework. Invarious embodiments 2, 3, 4, 5, 6, 7 or 8 or more proteins may becombined into a single construct. In various embodiments the 2, 3, 4, 5,6, 7 or 8 or more proteins may have the same sequence. It will beappreciated that either one of these approaches may require the proteinsto be chemically modified (e.g., to include pendant reactive groups)prior to coupling. It will also be appreciated that the multivalentcross-linking agents of the present disclosure are not limited to aparticular coupling reaction or framework (e.g., they can be preparedusing frameworks that include polymeric and/or non-polymericstructures). It will further be appreciated that the frameworks may belinear, branched, dendrimeric and/or a combination of these. Exemplaryframeworks and coupling chemistries are described below in the contextof the conjugates.

In various embodiments the monovalent binding proteins are covalentlylinked to each other or a framework. In such embodiments, the proteinscan be directly linked (i.e., with no intervening chemical groups) orindirectly linked through a spacer (e.g., a coupling agent or covalentchain that provides some physical separation between the protein orbetween the proteins and framework). As discussed below in the contextof the conjugates it is to be understood that proteins may be covalentlylinked to each other or a framework through any number of chemicallinkages, including but not limited to amide, ester, ether, isourea, andimine bonds.

In various embodiments, two or more monovalent binding proteins can benon-covalently linked to each other or to a framework. In certainembodiments, the dissociation constant (K_(d)) of the non-covalentlinkage in human serum is less than 1 pmol/L. For example, proteins maybe non-covalently linked to each other or a framework via a non-covalentligand-receptor pair as is well known in the art (e.g., withoutlimitation a biotin-avidin based pair). In such an embodiment, onemember of the ligand receptor-pair is covalently linked to one proteinwhile the other member of the pair is covalently linked to the otherprotein or framework. When the proteins (or proteins and framework) arecombined, the strong non-covalent interaction between the ligand and itsreceptor causes the proteins to become non-covalently linked to eachother (or the framework). Typical ligand/receptor pairs includeprotein/co-factor and enzyme/substrate pairs. Besides the commonly usedbiotin/avidin pair, these include without limitation,biotin/streptavidin, digoxigenin/anti-digoxigenin, FK506/FK506-bindingprotein (FKBP), rapamycin/FKBP, cyclophilin/cyclosporin andglutathione/glutathione transferase pairs. Other suitableligand/receptor pairs would be recognized by those skilled in the art,e.g., monoclonal antibodies paired with a epitope tag such as, withoutlimitation, glutathione-S-transferase (GST), c-myc, FLAG® and furtherthose described in Kessler pp. 105-152 of Advances in Mutagenesis” Ed.by Kessler, Springer-Verlag, 1990; “Affinity Chromatography: Methods andProtocols (Methods in Molecular Biology)” Ed. by Pascal Baillon, HumanaPress, 2000; and “Immobilized Affinity Ligand Techniques” by Hermansonet al., Academic Press, 1992.

2. Polynucleotide Cross-Linking Agents

In various embodiments, the multivalent cross-linking agents may includea polynucleotide aptamer. The polynucleotide aptamers bind the targetmolecule and are multivalent (i.e., capable of binding more than onetarget molecule). In general, monovalent aptamers will first begenerated based on their binding properties for the target molecule. Asis well known in the art, aptamers to a variety of target molecules canbe generated through a process of in vitro selection. See Ellington andSzostak (1990) Nature 346:818; Tuerk and Gold (1990) Science 249:505;and U.S. Pat. No. 5,582,981. See also the polynucleotide aptamers thatare described in U.S. Provisional Application No. 61/162,092 filed Mar.20, 2009 and corresponding PCT application filed Jan. 27, 2010, each ofwhich is incorporated herein by reference.

Typically, the process begins with the synthesis of a library consistingof randomly generated polynucleotide sequences of fixed length flankedby constant 5′ and 3′ ends that serve as primers. In certain embodiments(e.g., when optimizing an aptamer) one might start with a sequence whichis known to bind the target molecule and generate a library whichincludes a collection of polynucleotides which exhibit a limited rangeof changes from the starting sequence (e.g., a random set of singlemutations). The sequences in the library are then exposed to the targetmolecule and those that do not bind the target are removed (e.g., byaffinity chromatography). The bound sequences are then eluted andamplified (e.g., by cloning and subsequent transcription or by PCR) toprepare for subsequent rounds of selection in which the stringency ofthe elution conditions is increased or modified to identify sequenceswith the desired binding affinity and/or specificity. Jarosch et al.(2006) Nucleic Acids Res. 34:86 have described methods that allow theprocess to be performed without the constant primer regions.

In various embodiments, the selection process may involve steps in whichthe stringency of the elution conditions are gradually increased inorder to select aptamers with high affinity for the target molecule.

In various embodiments, the selection process may involve steps in whichthe elution conditions are modified (e.g., by using a different affinitycolumn) in order to select aptamers with desired specificity for thetarget molecule.

In various embodiments the selection process may generate a collectionof sublibraries (or “pools”) each of which comprises aptamers withsimilar affinities and/or specificities for the target molecule. Invarious embodiments the selection process may generate a single aptamersequence (or “monoclonal”). In various embodiments the aptamers are DNAbased. In various embodiments the aptamers are RNA based. In variousembodiments the aptamers are mixed RNA/DNA aptamers.

Multivalent aptamers can be generated by covalently or non-covalentlylinking two or more of these monovalent aptamers into a singleconstruct. An exemplary method is described in Example 4 below.Typically, two or more aptamers (which may have the same or differentsequences) may be bound directly to one another (e.g., via a couplingagent) or indirectly through an independent framework. In variousembodiments 2, 3, 4, 5, 6, 7 or 8 aptamers may be combined into a singleconstruct. In various embodiments the 2, 3, 4, 5, 6, 7 or 8 aptamers mayhave the same sequence. It will be appreciated that either one of theseapproaches may require the aptamers to be chemically modified (e.g., toinclude pendant reactive groups) prior to coupling. It will also beappreciated that the aptamers of the present disclosure are not limitedto a particular coupling reaction or framework (e.g., they can beprepared using frameworks that include polymeric and/or non-polymericstructures). It will further be appreciated that the frameworks may belinear, branched, hyperbranched and/or a combination of these. Exemplaryframeworks and coupling chemistries are described below in the contextof the conjugates.

In various embodiments the aptamers are covalently bound to each otheror a framework. In such embodiments, the aptamers can be directly bound(i.e., with no intervening chemical groups) or indirectly bound througha spacer (e.g., a coupling agent or covalent chain that provides somephysical separation between the aptamers or between the aptamers andframework). As discussed above in the context of the conjugates it is tobe understood that aptamers may be covalently bound to each other or aframework through any number of chemical linkages, including but notlimited to amide, ester, ether, isourea, and imine bonds.

In various embodiments, the two or more aptamers are non-covalentlybound to each other or to a framework. In certain embodiments, thedissociation constant (K_(d)) of the non-covalent linkage in human serumis less than 1 pmol/L. For example, aptamers may be non-covalently boundto each other or a framework via a non-covalent ligand-receptor pair asis well known in the art (e.g., without limitation a biotin-avidin basedpair). In such an embodiment, one member of the ligand receptor-pair iscovalently bound to one aptamer while the other member of the pair iscovalently bound to the other aptamer or framework. When the aptamers(or aptamers and framework) are combined, the strong non-covalentinteraction between the ligand and its receptor causes the aptamers tobecome non-covalently bound to each other (or the framework). Typicalligand/receptor pairs include protein/co-factor and enzyme/substratepairs. Besides the commonly used biotin/avidin pair, these includewithout limitation, biotin/streptavidin, digoxigenin/anti-digoxigenin,FK506/FK506-binding protein (FKBP), rapamycin/FKBP,cyclophilin/cyclosporin and glutathione/glutathione transferase pairs.Other suitable ligand/receptor pairs would be recognized by thoseskilled in the art, e.g., monoclonal antibodies paired with a epitopetag such as, without limitation, glutathione-S-transferase (GST), c-myc,FLAG® and further those described in Kessler pp. 105-152 of Advances inMutagenesis” Ed. by Kessler, Springer-Verlag, 1990; “AffinityChromatography: Methods and Protocols (Methods in Molecular Biology)”Ed. by Pascal Baillon, Humana Press, 2000; and “Immobilized AffinityLigand Techniques” by Hermanson et al., Academic Press, 1992.

3. Chemical Modification of Cross-Linking Agents

In general, it is to be understood that any of the aforementionedmultivalent cross-linking agents may be chemically modified, e.g., inorder to mitigate undesirable properties.

i. Non-Specific Modifications

In US 2007-0110811 we described the benefits of pegylating lectins inorder to reduce their in vivo mitogenicity. Thus, in certainembodiments, a multivalent cross-linking agent may be covalentlymodified with one or more compounds. Without limitation this mightinvolve reaction with an activated pegylation (PEG) agent (e.g., withoutlimitation N-hydroxysuccinimide activated PEG, succinimidyl ester of PEGpropionic acid, succinimidyl ester of PEG butanoic acid, succinimidylester of PEG alpha-methylbutanoate, etc.), another water soluble butnon-PEG-containing polymer such as poly(vinyl alcohol), a reagent thatcan be easily coupled to lysines, e.g., through the use of carbodiimidereagents, a perfluorinated compound, etc. The skilled artisan willreadily recognize other suitable compounds, e.g., by referring to thecomprehensive review that can be found in “Chemical Reagents for ProteinModification” by Lundblad, CRC Press, 3^(rd) Edition, 2004.

In general, the compound(s) may be attached to a multivalentcross-linking agent (e.g., a mitogenic lectin) via any of a number ofattachment methods known to those skilled in the art (e.g., via amine,carboxyl, hydroxyl or sulfhydryl groups). The potential covalentlinkages are similarly diverse (e.g., including amide bonds, carbamatebonds, ester bonds, thioether bonds, ether bonds, disulfide bonds,etc.). In certain embodiments suitable reactive groups can be graftedonto a multivalent cross-linking agent (e.g., a mitogenic lectin) byintroducing an appropriate amino acid by site-directed mutagenesis as isknown in the art. For example, PEGs are conveniently attached throughamino or carboxyl groups. Amino acid residues with free amino groupsinclude lysine residues and N-terminal amino acid residues. Amino acidresidues with free carboxyl groups include aspartic acid residues,glutamic acid residues and C-terminal amino acid residues. Sulfhydrylgroups found in cysteine residues may also be used as a reactive groupfor attaching the PEGs (or other compounds). In preferred embodimentsPEGs are covalently attached to an amino group, especially the freeamino group found in lysine residues.

Numerous methods for directly attaching PEGs to proteins are describedin Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304, 1992;Francis et al., Intern. J. of Hematol. 68:1-18, 1998; U.S. Pat. No.4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466. Onesuch method uses tresylated monomethoxy poly(ethylene glycol) (MPEG),which is produced by reacting MPEG with tresylchloride (ClSO₂CH₂CF₃).Tresylated MPEG reacts with exposed amine groups on lectins. A skilledperson will recognize that the invention is not limited to any specificpegylation agent (or compound) and will be able to identify othersuitable compounds that are known in the art.

In certain embodiments PEGs (or other compounds) may be attached to amultivalent cross-linking agent via an intervening linker. For example,U.S. Pat. No. 5,612,460, discloses urethane linkers for connecting PEGto proteins. PEGs can be attached to a protein via a linker by reactionwith compounds such as MPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional PEG derivatives and reaction chemistries for attachingPEG to proteins are described in WO 98/32466 and other patents, e.g.,those that are assigned to Shearwater of Huntsville, Ala.; NektarTherapeutics of San Carlos, Calif.; and/or Enzon Pharmaceuticals ofBridgewater, N.J. Catalogues can be obtained from these commercial PEGsuppliers that describe a range of suitable PEG compounds andchemistries (e.g., see the Nektar Advanced PEGylation CATALOG 2004).

In various embodiments, N-terminal alpha-amine and/or epsilon-aminolysine groups of polypeptide based cross-linking agents may besuccinylated and/or acetylated to change the charge distribution as wellas any tertiary and quaternary effects associated with such changes. Forexample, polypeptides may be succinylated by reaction in a saturatedsodium acetate buffer with an excess of succinic anhydride. Acetylationmay be performed using the same procedure but with acetic anhydride asthe modifying agent. For example, when the protein is concanavalin A,both acetylation and succinylation not only increase the density ofnegative charge within the polypeptide but also forces it to assemble asdimers instead of tetramers at physiological pH (e.g., see Agrawal etal., Biochemistry. 7:4211-4218, 1968 and Gunther et al., Proc. Natl.Acad. Sci. (USA) 70:1012-1016, 1973). In addition, the in vivo safetyprofile of these resulting materials is greatly improved as a result.

ii. Binding-Site Modifications

In certain embodiments, it may be advantageous to use an alternative andmore specific method for modifying the multivalent cross-linking agents.In particular, we have found that certain low molecular weightconjugates of the present disclosure do not form insoluble drug deliverysystems when combined with highly pegylated lectins made using highmolecular weight PEG reagents (>5 kDa). This poses a challenge since wehave previously found that lower molecular weight PEGs (<5 kDa) are muchless effective in reducing lectin mitogenicity. Without wishing to belimited to any particular theory, it may be that the larger PEG groupsare capable of sterically preventing binding and network formation withsmaller low-valency conjugates, but not larger high-valency conjugates.In view of this, we devised an alternative non-PEG based solution forimproving the safety profile of lectin-based cross-linking agents. Weachieved this by specifically targeting and modifying the sugar bindingsite of lectins. For example, by reacting a mannose ligand directly intothe concanavalin A binding site and purifying the unreacted material byhigh affinity ligand chromatography, we have been able to synthesizecross-linking agents with safety profiles that rival those of the bestpegylated lectins. Without wishing to be limited to any particulartheory, the functional concept appears to be that cell surfaces have adefined sugar affinity, valency, and ligand density, whereas theconjugates can have all of these properties adjusted by design. Thus,while incorporation of mannose into the lectin binding site completelyabolishes the cross-linking agents ability to bind and therebyagglutinate or stimulate cells, incorporation of a higher density ofhigher affinity ligands on the conjugates still allows gel formation. Incertain embodiments, incorporation of a small degree of pegylation withlow MW, discrete PEG chains may be used to stabilize the multivalentlectins in solution under a variety of extreme storage conditions,yielding manufacturable, safe, functional cross-linking agents whichcomplement the newly engineered conjugates.

In general, binding-site modified lectins will include at least onecovalently linked affinity ligand which is capable of associating withone of lectin binding sites. In various embodiments, the modifiedlectins may include just one covalently linked affinity ligand. Invarious embodiments, the lectins may include one covalently linkedaffinity ligand per binding site. Typically a multivalent lectin willinclude 2 or 4 binding sites (e.g., a dimer or tetramer of a monovalentlectin) but the present disclosure also encompasses lectins with 3, 5 ormore binding sites. The present disclosure also encompasses lectins withmore than one covalently linked affinity ligand per binding site. Thepresent disclosure further encompasses materials which include a mixtureof lectins that include different numbers of covalently linked affinityligands and/or that include unmodified lectins.

Any affinity ligand can be used for this purpose as long as it canassociate with a binding site of the lectin once covalently linked tothe lectin. Typically an affinity ligand will include a recognitionelement which interacts with the lectin binding site and a reactivelinker which enables the affinity ligand to become covalently attachedto the lectin once the recognition element is bound within the bindingsite.

Recognition Element

Any recognition element that can compete for binding with the lectin'scognate ligand (e.g., glucose or mannose in the case of Con A) could beused in an affinity ligand of the present disclosure. In variousembodiments, the recognition element includes a saccharide. In certainembodiments the saccharide is a natural saccharide (e.g., glucose,fructose, galactose, mannose, arabinose, ribose, xylose, etc.). Incertain embodiments the saccharide is a modified saccharide (e.g.,2′-fluororibose, 2′-deoxyribose, hexose, etc.). In certain embodimentsthe recognition element is glucose, sucrose, maltose, mannose,derivatives of these (e.g., glucosamine, mannosamine, methylglucose,methylmannose, ethylglucose, ethylmannose, etc.) and/or higher ordercombinations of these (e.g., linear and/or branched bimannose, linearand/or branched trimannose, etc.).

Other exemplary saccharides will be recognized by those skilled in theart. In particular, it is to be understood that depending on theapplication any one of the saccharides that are described above in thecontext of the conjugate affinity ligands may be used (e.g., any one ofthe saccharides of formula IVa or IVb). In certain embodiments, therecognition element includes a monosaccharide. In certain embodiments,the recognition element includes a disaccharide. In certain embodiments,the recognition element includes a trisaccharide. In some embodiments,the recognition element includes a saccharide and one or more aminegroups. In some embodiments, the recognition element isaminoethylglucose (AEG). In some embodiments, the recognition element isaminoethylmannose (AEM). In some embodiments, the recognition element isaminoethylbimannose (AEBM). In some embodiments, the recognition elementis aminoethyltrimannose (AETM). In some embodiments, the recognitionelement is (β-aminoethyl-N-acetylglucosamine (AEGA). In someembodiments, the recognition element is aminoethylfucose (AEF). In otherembodiments, the recognition element is D-glucosamine (GA).

In various embodiments, the recognition element includes apolysaccharide, glycopeptide or glycolipid. In certain embodiments, therecognition element includes from 2-10 saccharide moieties, e.g., 2, 3,4, 5, 6, 7, 8, 9 or 10 moieties. The terminal and/or internal residuesof the polysaccharide, glycopeptide or glycolipid may be selected basedon the saccharide specificity of the lectin in question (e.g., seeGoldstein et al., Biochem. Biophys. Acta 317:500-504, 1973 and Lis etal., Ann. Rev. Biochem. 55:35-67, 1986).

In various embodiments, the recognition element for a particularlectin/exogenous target molecule combination may be selectedempirically. According to such embodiments one or more recognitionelements are screened based on their relative binding affinities for thelectin as compared to the exogenous target molecule. In certainembodiments a library of saccharides and/or polysaccharides are screenedin this manner. A suitable recognition element will exhibit a detectablelevel of competition with the exogenous target molecule but will notcompete so strongly that it prevents all binding between the lectin andthe exogenous target molecule. In certain embodiments, differentrecognition elements may be screened by testing the effect of differentaffinity ligands on relevant lectin properties (e.g., based on theirability to inhibit agglutination and/or their material set points asdiscussed in more detail below and in the Examples). In certainembodiments, the recognition element will be selected in view of theconjugate that the modified lectin is to be combined with (e.g., so thatthe conjugate is able to displace the recognition element from thebinding site and thereby form a cross-linked material).

Reactive Linker

Affinity ligands may be covalently linked to a lectin in any manner.Most methods will involve allowing the recognition element of the ligandto associate with the lectin binding site and then causing the reactivelinker to react with the lectin. In certain embodiments, the reactivelinker may be attached to the recognition element at a position thatdoes not substantially interfere with the binding properties of therecognition element. For example, when the recognition element is asaccharide or polysaccharide the linker may be attached to the C1, C2 orC6 position of a terminal saccharide. In certain embodiments, the linkermay be attached to the C1 position. The C1 position is also referred toas the anomeric carbon and may be connected to the linker in the alphaor beta conformation. In certain embodiments, the linker is attached tothe C1 position as the alpha anomer.

In certain embodiments, photoactivatable linkers may be used. Forexample, Beppu et al., J. Biochem. 78:1013-1019, 1975, described amethod in which an arylazido linker was activated using ultravioletlight to form a covalent bond between concanavalin A and a sugarderivative within the binding site. Similar results were recorded byFraser et al., Proc. Natl. Acad. Sci. (USA) 73:790-794, 1976 usingsuccinylated concanavalin A. A similar procedure has also been employedusing ricin and a photoactivatable derivative of galactose as describedby Houston, J. Biol. Chem. 258:7208-7212, 1983. Photoactivatablederivatives of complex glycopeptide ligands having a higher affinity forlectins than saccharides and disaccharides have also been described byBaenziger et al., J. Biol. Chem. 257:4421-4425, 1982. These derivativeswere made by covalently linking a photoactivatable group to the peptideportion of the glycopeptide ligand.

In general, any photoactivatable linker may be used such as an aryl,purine, pyrimidine, or alkyl azide, a diazo or diazirine group, abenzophenone, or a nitrobenzene. A more comprehensive list ofpotentially useful photoactivatable linkers may be found in Fleming,Tetrahedron 51:12479-12520, 1995 as well as Brunner, Annu. Rev. Biochem.62:483-514, 1993 and Wong, S. S. “Chemistry of Protein Conjugation andCross-Linking”, (1993), CRC Press, New York, pp. 168-194.

In various embodiments, the photoactivatable linker may include adiazirine group. Photoactivation of diazirine groups with ultraviolet(UV) light creates reactive carbene intermediates that can form covalentbonds through addition reactions with any amino acid side chain orpeptide backbone within range of the linker. Long wavelength UV-light(about 320-370 nm, preferably about 345 nm) is typically used toactivate diazirines (e.g., see Suchanek et al., Nat. Methods 2:261-268,2005).

In various embodiments, the photoactivatable linker may include an arylazide group. When aryl azide groups are exposed to UV-light they formnitrene groups that can initiate addition reactions with double bonds,insertion into C—H and N—H sites, or subsequent ring expansion to reactas a nucleophile with primary amines. The latter reaction pathpredominates when primary amines are present in the sample. Withoutlimitation, long wavelength UV-light (about 320-370 nm, preferably about366 nm) is thought to be most efficient for substituted aryl azides(e.g., with hydroxy or nitro groups) while shorter wavelengths arethought to be most efficient for unsubstituted aryl azides. SuitableUV-light sources are available commercially, e.g., from Pierce,Rockford, Ill.

For example, in various embodiments the affinity ligand may be of thegeneral formula (IX): R_(e)-L¹ where R_(e) is a recognition element and-L¹ is a reactive linker. In certain embodiments R_(e) is a saccharidemoiety. In certain embodiments R_(e) is a glucose or mannose moietywhich is covalently bonded to the linker at the C1 position.

In certain embodiments -L¹ may be of the general formula (Xa):

wherein:

R³ is independently selected from the group consisting of hydrogen, —OH,—NO₂, and halogen (e.g., —F or —Cl);

X^(L) is a covalent bond or a bivalent, straight or branched, saturatedor unsaturated, optionally substituted C₁₋₂₀ hydrocarbon chain whereinone or more methylene units of X^(L) are optionally and independentlyreplaced by —O—, —S—, —N(R′)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R′)C(O)—,—C(O)N(R′)—, —S(O)—, —S(O)₂—, —N(R′)SO₂—, —SO₂N(R′)—, a heterocyclicgroup, an aryl group, or a heteroaryl group; and

each occurrence of R′ is independently hydrogen, a suitable protectinggroup, or an acyl moiety, arylalkyl moiety, aliphatic moiety, arylmoiety, heteroaryl moiety, or heteroaliphatic moiety.

In any case where a chemical variable is shown attached to a bond thatcrosses a bond of ring (for example as shown for R³ above), this meansthat one or more such variables are optionally attached to the ringhaving the crossed bond. Each R³ group on such a ring can be attached atany suitable position; this is generally understood to mean that thegroup is attached in place of a hydrogen atom on the parent ring. Thisincludes the possibility that two R³ groups can be attached to the samering atom. Furthermore, when more than one R³ group is present on aring, each may be the same or different than other R³ groups attachedthereto, and each group is defined independently of other groups thatmay be attached elsewhere on the same molecule, even though they may berepresented by the same identifier.

In certain embodiments, the —N₃ group is in the meta position. Incertain embodiments, the —N₃ group is in the ortho position. In certainembodiments, the —N₃ group is in the para position.

In certain embodiments, one, two, three, four, or five methylene unitsof X^(L) are optionally and independently replaced. In certainembodiments, X^(L) is constructed from a C₁₋₁₀, C₁₋₈, C₁₋₆, C₁₋₄, C₂₋₁₂,C₄₋₁₂, C₆₋₁₂, C₈₋₁₂, or C₁₀₋₁₂ hydrocarbon chain wherein one or moremethylene units of X^(L) are optionally and independently replaced by—O—, —S—, —N(R′)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R′)C(O)—, —C(O)N(R′)—,—S(O)—, —S(O)₂—, —N(R′)SO₂—, —SO₂N(R′)—, a heterocyclic group, an arylgroup, or a heteroaryl group. In some embodiments, one or more methyleneunits of X^(L) is replaced by a heterocyclic group. In some embodiments,one or more methylene units of X^(L) is replaced by a triazole moiety.In certain embodiments, one or more methylene units of X^(L) is replacedby —C(O)—. In certain embodiments, one or more methylene units of X^(L)is replaced by —C(O)N(R′)—. In certain embodiments, one or moremethylene units of XL is replaced by —O—.

In some embodiments, X^(L) is

In some embodiments, X^(L) is

In some embodiments, X^(L) is

In some embodiments, X^(L) is

In some embodiments, X^(L) is

In some embodiments, X^(L) is

In certain embodiments -L¹ may be of the general formula (Xb):

where X^(L) is as defined above for formula Xa; and

R⁴ is hydrogen, C₁-C₆ alkyl or —CF₃.

In certain embodiments, non-photoactivatable linkers may be used. Forexample, U.S. Pat. Nos. 5,239,062 and 5,395,924 describe linkers thatcan be activated by changes in pH or temperature. Exemplary reactivelinkers which are discussed include those which can be introduced intoan affinity ligand using reagents such as cyanuric chloride (Kay et al.,Nature 216:514-515, 1967) or dichloro-S-triazines such as2-amino-4,6-dichloro-S-triazine (Kay et al., Biochim. Biophys. Acta198:276-285, 1970) and 2,4-dichloro-6-methoxy-S-triazine (Lang et al.,J. Chem. Soc. Perkin 1:2189-2194, 1977). Reactive linkers withNHS-esters or aldehydes that would react primarily with terminal aminessuch as those found on lysines could also be used.

In various embodiments, the reactive linker for a particularlectin/target molecule combination may be selected empirically.According to such embodiments several affinity ligands with the samerecognition element and different linkers (e.g., linkers of differentlengths, linkers with different reactive groups, linkers with differenthydrophobicity, etc.) are screened based on their effect on relevantlectin properties (e.g., based on their ability to inhibit agglutinationand/or their material set points as discussed in more detail below andin the Examples).

ii. Extent of Modification

In general, the number of compounds that are attached to eachmultivalent cross-linking agent (i.e., the degree of substitution) willvary based on the nature of the cross-linking agent, the nature of thecompound(s), the number of reaction sites available and the reactionconditions. For example, the subunits of concanavalin A each includetwelve lysine residues. As a result, if concanavalin A is pegylated witha compound that reacts with lysine residues, then each subunit could becovalently linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of thesecompounds. Conversely, each subunit of concanavalin A includes just oneglucose binding site. Thus, if concanavalin A is reacted with a compoundthat reacts at the binding site, then each subunit will be covalenlylinked to just one such compound. Methods for determining the degree ofsubstitution are discussed in Delgado et al., Crit. Rev. Thera. DrugCarrier Sys. 9:249-304, 1992.

In preferred embodiments, the chemical modification of a multivalentcross-linking agent may be optimized using a plurality of compounds anda plurality of reaction conditions (e.g., that vary the reagentconcentrations, pH, temperature, etc.). Preferred compounds and reactionconditions are such that desirable properties (e.g., binding affinity)are not substantially impaired while undesirable properties (e.g.,mitogenicity) are reduced as compared to an unmodified cross-linkingagent. For example, an automated robotic handling device may be used toprepare a range of modified compositions with different compounds anddifferent reaction conditions. Using routine orthogonal experimentationa skilled person can then screen the properties of the treatedcompositions. In certain embodiments further rounds of orthogonaloptimization are performed around the preferred conditions to furtherrefine the preferred compounds and reaction conditions.

In one embodiment, optimal reaction conditions are identified byseparating treated compositions by electrophoresis, preferably bydenaturing SDS-PAGE electrophoresis. In various embodiments,compositions which include uniformly modified cross-linking agents arepreferred. These preferred compositions will have weaker bands at themolecular weight of the unmodified cross-linking agent as measured bySDS-PAGE.

4. Purification of Cross-Linking Agents

In various embodiments, multivalent cross-linking agents (whether theyhave been chemically modified or not) can be further processed in orderto improve their properties. Thus, in certain embodiments, compositionscomprising multivalent cross-linking agents can be purified in order toremove protein fragments, unmodified components, etc. In general, theseseparations can be achieved on the basis of physical properties (e.g.,electrical charge; molecular weight; and/or size) and/or chemicalproperties (e.g., binding affinity for a target molecule). In certainembodiments optimal removal may be achieved by combining two or moremethods that rely on these differential properties. In one embodiment,these separations are performed under denaturing conditions. Forexample, unmodified or partially modified cross-linking agents can beremoved on the basis of their net charge by ion-exchange chromatography.Gel-filtration chromatography may be used to discriminate betweendifferentially modified cross-linking agents on the basis of size.Affinity chromatography is another method that may be used to removeunmodified or partially modified cross-linking agents. This approachtakes advantage of the differential binding affinity of modified,partially modified and unmodified cross-linking agents for a specifictarget molecule.

5. Characterization of Cross-Linking Agents

In various embodiments, multivalent cross-linking agents (whether theyhave been chemically modified or not) can be screened or further testedin order to confirm or characterize their properties. Representativeassays include: affinity assays, agglutination assays, T-cellmitogenicity assays, T-cell viability assays, antigenicity assays, etc.

Affinity assays may involve passing the multivalent cross-linking agentover an affinity column (e.g., a resin with the target molecule) anddetermining the elution conditions required to remove the cross-linkingagent from the column. Equilibrium dialysis can also be used as is knownin the art. Set point assays in which the cross-linking agent iscombined with one or more conjugates of the present disclosure and thencontacted with varying concentrations of the target molecule may also beused. Preferably the binding affinity of a chemically modifiedcross-linking agents is at least 75% that of the unmodifiedcross-linking agent. More preferably the binding affinity is at least85% and yet more preferably at least 95% that of the unmodifiedcross-linking agent.

In certain embodiments, an agglutination assay may be used to determinethe minimum agglutinating concentration (MAC) of a multivalentcross-linking agent. For example, in certain embodiments the MAC may bedetermined using rabbit erythrocytes as described in US 20070110811. Wehave found that higher MAC values correlate strongly with reducedmitogenicity in the case of chemically modified lectins. In certainembodiments a modified cross-linking agent may have a MAC that is higherthan the unmodified cross-linking agent. Preferably the MAC is 25 timesthat of the unmodified cross-linking agent. More preferably the MAC is50 times and yet more preferably more than 100 times that of theunmodified cross-linking agent. In certain embodiments, the modifiedcross-linking agent exhibits a MAC with a 2% v/v suspension offormaldehyde-stabilized rabbit erythrocytes that is greater than 4ug/ml. Preferably the MAC is greater than 6 ug/ml, more preferablygreater than 10 ug/ml, even more preferably greater than 25 ug/ml.

Mitogenicity assays will typically involve contacting the compositionsof interest with a T-cell culture (e.g., PBMC cells) for a period oftime and then measuring the level of T-cell proliferation. Variousmethods for measuring cell proliferation are known. In one embodimentthe cell density may be measured spectrophotometrically at 450 nm. Inanother embodiment an indirect measure can obtained by detecting thereduction of MTT at 570 nm (e.g., see Ohno et al., J. Immunol. Methods145:199-203, 1991). In preferred embodiments, the level of cellproliferation is determined using a tritiated thymidine uptake assay.Those skilled in the art will recognize that other suitable methods maybe used and that the invention is in no way limited to a specificproliferation assay. In certain embodiments, the T-cell mitogenicity ofa modified cross-linking agent is less than 50% the T-cell mitogenicityof the unmodified cross-linking agent. The reduction in T-cellmitogenicity may be assessed by performing a comparative thymidineuptake assay across a range cross-linking agent concentrations, e.g.,0.01, 0.1, 1, 10, 100 and 1000 ug/ml. In preferred embodiments, thethymidine uptake assay is performed with samples that includeapproximately 500,000 PBMCs. The mitogenicity of the test composition(e.g., a modified composition) is then expressed as the % maximalunmodified mitogenicity. The % maximal unmodified mitogenicity isobtained by dividing the maximal CPM (counts per minute) value for thetest composition over all measured concentrations by the maximal CPMvalue of the unmodified composition over all measured concentrations.Preferably, the test composition with reduced mitogenicity induces alevel of T-cell proliferation that is at least 50% lower than theunmodified composition. More preferably, the level is at least 75%lower, even more preferably at least 90%, 95% or 99% lower.

T-cell viability can be measured using a similar experiment by addingTrypan Blue to the T-cell culture and counting a representative sampleof the cells (noting those that either take up the trypan or stillexclude the trypan, i.e., those that become blue vs. those that do not).The % viability is then calculated by dividing the number of cells thatexclude the trypan (alive, “not blue”) by the total number of cellscounted (dead, “blue,” plus live, “not blue”). Those skilled in the artwill recognize that other suitable methods may be used and that theinvention is in no way limited to a specific viability assay. In certainembodiments, a modified cross-linking agent exhibits a percentage cellviability at 100 ug/ml that is greater than 10% when assayed using PBMCsat a concentration of 500,000 cells/ml. Preferably the percentage cellviability is greater than 25%, more preferably greater than 50%, evenmore preferably greater than 90%.

Cross-Linked Materials

When cross-linking agents and conjugates are combined in the absence ofthe exogenous target molecule, a non-covalently cross-linked material isformed. In various embodiments, the material may be prepared in aqueoussolution through self-assembly by mixing solutions of the cross-linkingagent and conjugate. In various embodiments, particles of the materialmay be prepared by reverse emulsion. As described in more detail in US2004/0202719, this can be achieved by adding the aforementioned aqueoussolution to a mixture of a hydrophobic liquid and a surfactant andagitating the mixture.

Once formed, the cross-linked material can be used for a variety ofapplications. When the material is placed in the presence of freeexogenous target molecules these compete for the interactions betweenthe cross-linking agents and the conjugates. Above a certainconcentration of free exogenous target molecule, the level ofcompetition becomes such that the material begins to degrade byreleasing conjugates from the surface. In various embodiments, theextent and/or rate of release increases as the concentration ofexogenous target molecule increases. As a result, conjugates arereleased from the material in a manner which is directly tied to thelocal concentration of the exogenous target molecule.

In general, the release properties of the material will depend on thenature of the cross-linking agents, conjugates, exogenous targetmolecule and conditions (e.g., pH, temperature, nature and concentrationof endogenous molecules that bind the cross-linking agent, etc.). If theaffinity of the cross-linking agents for the conjugates is much greaterthan for the exogenous target molecule then the material will onlyrelease conjugates at high concentrations of exogenous target molecule.As the relative affinity of the cross-linking agents for the conjugatesis decreased, release of conjugates from the material will occur atlower exogenous target molecule concentrations. The release propertiesof the material can also be adjusted by varying the relative amounts ofcross-linking agent to conjugate. Higher ratios of cross-linking agentto conjugate will lead to materials that release conjugates at higherexogenous target molecule concentrations. Lower ratios of cross-linkingagent to conjugate will lead to materials that release conjugates atlower exogenous target molecule concentrations. It will be appreciatedthat, depending on the application, these variables will enable one toproduce materials which respond to a wide variety of exogenous targetmolecule concentrations.

In various embodiments, the cross-linked material is insoluble whenplaced in pH 7 HEPES buffered saline at 37 C (25 mM HEPES containing 150mM NaCl). In various embodiments, the cross-linked material remainssubstantially insoluble when exogenous target molecule is added to thebuffer up to a threshold concentration called the set point. Above theset point, the cross-linked material exhibits an increase in the extentand rate of release of conjugates. It will be appreciated that thistransition may occur sharply or may occur gradually over a range ofconcentrations around the set point. In general, the desired set pointand transition will depend on the nature of the exogenous targetmolecule and the intended application for the material. In particular,when the material is designed to respond to an increase in the level ofa particular exogenous target molecule, the desired set point may bedetermined based on the PK profile of the exogenous target molecule (inparticular the C_(max)). It is to be understood that the amount ofexogenous target molecule present in a patient will depend on the route,dose and schedule of administration and further on the delivery means(e.g., immediate release, extended release, and/or delayed releaseformulations could be used for an orally delivered exogenous targetmolecule).

It will be appreciated that the desired set point for any exogenoustarget molecule can be readily determined for a variety of differentapplications. It will also be appreciated that the set point may need tobe adjusted for certain patients (e.g., based on patient gender,patients with abnormally low or high levels of absorption of theexogenous target molecule, etc.) or applications (e.g., a drug deliverysystem designed to release on a more frequent basis may require a lowerthreshold concentration than a system designed to release lessfrequently).

It will be appreciated that a material having a desired set point may begenerated via routine experimentation using the materials and methodsdescribed herein. For example, the same cross-linking agent andconjugate can be combined to produce a series of materials with agradually increasing ratio of cross-linking agent to conjugate (w/w).These materials will cover a spectrum of set points. Once a leadmaterial with a suitable set point has been identified the process canbe repeated with a finer resolution to yield an optimized material.Alternatively (or additionally) the same conjugate can be combined witha plurality of different cross-linking agents that have graduallyincreasing affinities for the conjugate. This will yield a plurality ofmaterials with a spectrum of set points that can be further refined(e.g., by varying the w/w ratio of cross-linking agent to conjugate).Alternatively one could initiate the process by combining the samecross-linking agent with a plurality of different conjugates. In variousembodiments, the conjugates may have varying affinities for thecross-linking agent (e.g., as a result of including different affinityligands). In various embodiments, the conjugates may include the sameaffinity ligands but have different molecular weights (e.g., as a resultof different conjugate frameworks).

In various embodiments, the material remains substantially insolublewhen placed at 37 C in normal human serum for six hours using USPdissolution test method II at 50 rpm. In various embodiments, less than1, 2, 4, 6, 8, or 10% of the material dissolves when placed at 37 C innormal human serum for six hours using USP dissolution test method II at50 rpm. In various embodiments, a material of the present disclosure mayremain substantially insoluble when placed in pH 7 HEPES buffered salinecontaining 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350or 400 mg/dL glucose at 37 C for six hours using USP dissolution testmethod II at 50 rpm. In various embodiments, less than 1, 2, 4, 6, 8, or10% of the material dissolves when placed in pH 7 HEPES buffered salinewith 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 or 400mg/dL glucose at 37 C for six hours using USP dissolution test method IIat 50 rpm.

Uses

In another aspect, the present disclosure provides methods of using thematerials. In general, the materials can be used to controllably releaseconjugates in response to an exogenous target molecule.

In various embodiments, a material may be used to controllably deliver adrug to a patient. The invention encompasses treating a disease orcondition by administering a material of the present disclosure.Although the materials can be used to treat any patient (e.g., dogs,cats, cows, horses, sheep, pigs, mice, etc.), they are most preferablyused in the treatment of humans. A material can be administered to apatient by any route. In general the most appropriate route ofadministration will depend upon a variety of factors including thenature of the disease or condition being treated, the nature of thedrug, the nature of the exogenous target molecule, the condition of thepatient, etc. In general, the present disclosure encompassesadministration by oral, intravenous, intramuscular, intra-arterial,subcutaneous, intraventricular, transdermal, rectal, intravaginal,intraperitoneal, topical (as by powders, ointments, or drops), buccal,or as an oral or nasal spray or aerosol. General considerations in theformulation and manufacture of pharmaceutical compositions for thesedifferent routes may be found, for example, in Remington'sPharmaceutical Sciences, 19^(th) ed., Mack Publishing Co., Easton, Pa.,1995.

In various embodiments, the material may be administered subcutaneously,e.g., by injection. The material can be dissolved in a carrier for easeof delivery. For example, the carrier can be an aqueous solutionincluding, but not limited to, sterile water, saline or buffered saline.In general, a therapeutically effective amount of a drug in the form ofa conjugate will be administered. By a “therapeutically effectiveamount” of a drug is meant a sufficient amount of the drug to treat(e.g., to ameliorate the symptoms of, delay progression of, preventrecurrence of, delay onset of, etc.) the disease or condition at areasonable benefit/risk ratio, which involves a balancing of theefficacy and toxicity of the drug. In general, therapeutic efficacy andtoxicity may be determined by standard pharmacological procedures incell cultures or with experimental animals, e.g., by calculating theED₅₀ (the dose that is therapeutically effective in 50% of the treatedsubjects) and the LD₅₀ (the dose that is lethal to 50% of treatedsubjects). The ED₅₀/LD₅₀ represents the therapeutic index of the drug.Although in general drugs having a large therapeutic index arepreferred, as is well known in the art, a smaller therapeutic index maybe acceptable in the case of a serious disease or condition,particularly in the absence of alternative therapeutic options. Ultimateselection of an appropriate range of doses for administration to humansis determined in the course of clinical trials.

In various embodiments, the drug is insulin and the average daily doseof insulin is in the range of 10 to 200 U, e.g., 25 to 100 U (where 1Unit of insulin is ˜0.04 mg). In certain embodiments, an amount ofmaterial with these insulin doses is administered on a daily basis. Incertain embodiments, an amount of material with 5 to 10 times theseinsulin doses is administered on a weekly basis. In certain embodiments,an amount of material with 10 to 20 times these insulin doses isadministered on a bi-weekly basis. In certain embodiments, an amount ofmaterial with 20 to 40 times these insulin doses is administered on amonthly basis. Those skilled in the art will be recognize that this sameapproach may be extrapolated to other approved drugs with known doseranges, e.g., any of the approved insulin sensitizers and insulinsecretagogues described herein.

It will be understood that the total daily usage of a drug for any givenpatient will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective amountfor any particular patient will depend upon a variety of factorsincluding the disease or condition being treated; the activity of thespecific drug employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration and rate of excretion of thespecific drug employed; the duration of the treatment; drugs used incombination or coincidental with the specific drug employed; and likefactors well known in the medical arts. In various embodiments, amaterial of the present disclosure may be administered on more than oneoccasion. For example, the present disclosure specifically encompassesmethods in which a material is administered by subcutaneous injection toa patient on a continuous schedule (e.g., once a day, once every twodays, once a week, once every two weeks, once a month, etc.).

In certain embodiments, a material of the present disclosure may be usedto treat hyperglycemia in a patient (e.g., a mammalian patient). Incertain embodiments, the patient is diabetic. However, the presentmethods are not limited to treating diabetic patients. For example, incertain embodiments, a material may be used to treat hyperglycemia in apatient with an infection associated with impaired glycemic control. Incertain embodiments, a material may be used to treat diabetes.

In various embodiments, a material of the present disclosure may beadministered to a patient who is receiving at least one additionaltherapy. In various embodiments, the at least one additional therapy isintended to treat the same disease or disorder as the administeredmaterial. In various embodiments, the at least one additional therapy isintended to treat a side-effect of the primary drug. The two or moretherapies may be administered within the same, overlapping ornon-overlapping timeframes as long as there is a period when the patientis receiving a benefit from both therapies. The two or more therapiesmay be administered on the same or different schedules as long as thereis a period when the patient is receiving a benefit from both therapies.The two or more therapies may be administered within the same ordifferent formulations as long as there is a period when the patient isreceiving a benefit from both therapies. In certain embodiments, asingle material of the present disclosure may include more than one drugfor treating the same disease or disorder. In certain embodiments, twoor more separate materials of the present disclosure may be administered(as a mixture or separately) that include different drugs for treatingthe same disease or disorder. In certain embodiments, an unconjugatedsecondary drug may be included in a material of the present disclosure(i.e., a drug which is simply mixed with the components of the materialand not covalently bound to the cross-linked material). For example, incertain embodiments, any of these approaches may be used to administermore than one anti-diabetic drug to a subject. Certain exemplaryembodiments of this inventive approach are described in more detailbelow in the context of insulin-related therapies; however, it will beappreciated from the foregoing that other therapies will benefit fromsuch combination approaches.

Insulin sensitizers (e.g., biguanides such as metformin, glitazones) actby increasing a patient's response to a given amount of insulin. Apatient receiving an insulin sensitizer will therefore require a lowerdose of an insulin-based material of the present disclosure than anotherwise identical patient would. Thus, in certain embodiments, amaterial comprising insulin conjugates may be administered to a patientwho is also being treated with an insulin sensitizer. In variousembodiments, the material of the present disclosure may be administeredat up to 75% of the normal dose required in the absence of the insulinsensitizer. In various embodiments, up to 50, 40, 30 or 20% of thenormal dose may be administered.

Insulin resistance is a disorder in which normal amounts of insulin areinadequate to produce a normal insulin response. For example,insulin-resistant patients may require high doses of insulin in order toovercome their resistance and provide a sufficient glucose-loweringeffect. In these cases, insulin doses that would normally inducehypoglycemia in less resistant patients fail to even exert aglucose-lowering effect in highly resistant patients. Similarly, thematerials of the present disclosure are only effective for this subclassof patients when they release high levels of insulin-conjugates in asuitable timeframe. In certain embodiments, the treatment of thissubclass of patients may be facilitated by combining the two approaches.Thus in certain embodiments, a traditional insulin-based therapy is usedto provide a baseline level of insulin and a material of the presentinvention is administered to provide a controlled supplement of insulinwhen needed by the patient. Thus, in certain embodiments, a materialcomprising insulin conjugates may be administered to a patient who isalso being treated with insulin. In various embodiments, the insulin maybe administered at up to 75% of the normal dose required in the absenceof the material of the present disclosure. In various embodiments, up to50, 40, 30 or 20% of the normal dose may be administered. It will beappreciated that this combination approach may also be used with insulinresistant patients who are receiving an insulin secretagogue (e.g., asulfonylurea, GLP-1, exendin-4, etc.) and/or an insulin sensitizer(e.g., a biguanide such as metformin, a glitazone).

Once the material has been administered as described above it can betriggered by administration of a suitable exogenous target molecule. Incertain embodiment, a triggering amount of the exogenous target moleculeis administered. As used herein, a “triggering amount” of exogenoustarget molecule is an amount sufficient to cause release of some amountof conjugate from the previously administered material. It is to beunderstood that any of the aforementioned methods of administration forthe material apply equally to the exogenous target molecule. It is alsobe to be understood that the methods of administration for the materialand exogenous target molecule may be the same or different. In variousembodiments, the methods of administration are different (e.g., forpurposes of illustration the material may be administered bysubcutaneous injection on a weekly basis while the exogenous targetmolecule is administered orally on a daily basis). The oraladministration of an exogenous target molecule is of particular valuesince it facilitates patient compliance. In general, it will beappreciated that the conjugate release profile from the material will berelated to the PK profile of the exogenous target molecule. Thus, theconjugate release profile can be tailored by controlling the PK profileof the exogenous target molecule. As is well known in the art, the PKprofile of the exogenous target molecule can be tailored based on thedose, route, frequency and formulation used. For example, if a short andintense release of conjugate is desired then an oral immediate releaseformulation might be used. In contrast, if a longer less intense releaseof conjugate is desired then an oral extended release formulation mightbe used instead. General considerations in the formulation andmanufacture of immediate and extended release formulation may be found,for example, in Remington's Pharmaceutical Sciences, 19^(th) ed., MackPublishing Co., Easton, Pa., 1995. In general, it will be appreciatedthat the set point of the material will be below the C_(max) of theexogenous target molecule formulation for conjugate release to occur.For example, in various embodiments, the set point may be less than 10,20, 30, 40, 50, 60, 70, 80 or 90% of the C_(max).

It will also be appreciated that the relative frequency ofadministration of a material of the present disclosure and an exogenoustarget molecule may be the same or different. In certain embodiments,the exogenous target molecule is administered more frequently than thematerial. For example, in certain embodiment, the material may beadministered daily while the exogenous target molecule is administeredmore than once a day. In certain embodiment, the material may beadministered twice weekly, weekly, biweekly or monthly while theexogenous target molecule is administered daily. In certain embodiments,the material is administered monthly and the exogenous target moleculeis administered twice weekly, weekly, or biweekly.

Kits

In another aspect the present disclosure provides kits that includecross-linking agents and conjugates and other reagents for preparing amaterial. For example, a kit may include separate containers thatinclude a plurality of conjugates and a plurality of cross-linkingagents. When the conjugates and cross-linking agents of the kit aremixed a cross-linked material is formed. In various embodiments, thematerial is designed for subcutaneous delivery and the kit includes asyringe. In various embodiments, a kit may include a syringe which ispre-filled with a cross-linked material. The kit may also includeinstructions for mixing the conjugates and cross-linking agents toproduce the cross-linked material. The kit may also include aformulation of the exogenous target molecule, e.g., an oral dosage formsuch as a capsule or tablet.

In yet another aspect, the present disclosure provides libraries ofconjugates and/or cross-linking agents. These libraries may beparticularly useful for generating materials with a desired set point.In various embodiments, a library may include a plurality ofcross-linking agents which produce different set points with the sameconjugate. In various embodiments, a library may further include one ormore conjugates which form cross-linked materials with cross-linkingagents in the library. When the library includes more than one suchconjugate, the different conjugates may have different molecularweights, a different number of affinity ligands per conjugate moleculeand/or different affinity ligands. In various embodiments, a library mayinclude one or more of the conjugates that include more than one type ofaffinity ligand. In various embodiments, a library may include aplurality of conjugates which produce different set points with the samecross-linking agents. In various embodiments, a library may furtherinclude one or more cross-linking agents which form cross-linkedmaterials with conjugates in the library.

Examples Example 1 Synthesis of α-Methyl-Mannose Triggered Material

An exemplary conjugate was synthesized according to the method inExample 8 using TSB-C4 as the scaffold, AEBM as the affinity ligand, andNH₂—B1-BOC2(A1,B29)-insulin as the drug (see FIGS. 1-2 for affinityligand and conjugate structure and Examples 3-7 for methods used toprepare these starting materials). 0.50 ml of a 2.3 mg/ml solution ofconjugate in pH 8.2, 25 mM HEPES buffer containing 0.150 M sodiumchloride (S14 buffer) was added to a centrifuge tube and subsequentlymixed rapidly with 0.500 ml of a 18 mg/ml native Con A (NCA) solution inpH 7.4, 25 mM HEPES buffer containing 0.150 M sodium chloride (S24buffer) to form a dispersion of insoluble particles. The dispersion wasallowed to sit at room temperature for 20 min and then separated fromthe supernatant by centrifugation. The resulting cake was washed 5× with1.0 ml of pH 7.4, 25 mM HEPES buffer containing 0.150 M sodium chloride(S24 buffer). After the last wash, the remaining insoluble material wasincubated overnight at 37 C. The next day, the remaining particles wereagain isolated by centrifugation and washed one additional time in 1.0ml of S24. The resulting insoluble material was dispersed in a totalvolume of 0.30 ml using S24 and set aside for future studies. Thisprocess may be scaled up directly to produce any amount of desiredproduct.

Example 2 α-Methyl-Mannose Triggering in Non-Diabetic Rats

0.300 ml of the formulation prepared in Example 1 was injectedsubcutaneously into each of three normal male Sprague Dawley (SD) rats(Charles River Laboratories, Wilmington, Mass.) weighing between 400 and500 g. Prior to formulation injection, blood glucose values weremeasured via tail vein bleeding using a Precision Xtra glucometer(Abbott Laboratories, Alameda, Calif.) and approximately 100 ul of serumwas obtained via tail vein bleeding to assay for background insulinlevels. Food was removed from the rat cages during the duration of thestudy. Serum and blood glucose values were obtained at 30 min, 60 min,90 min, and 120 min post-injection. At 120 min after the injection, anintraperitoneal injection of a 25% w/v α-methyl-mannose solution wasinjected to provide a 2 g/kg dose after which serum and blood glucosevalues were obtained at 135 min, 150 min, 180 min, 210 min, 240 min, and300 min. Serum insulin concentrations were subsequently measured with acommercially available ELISA kit (Human Insulin ELISA, Mercodia,Uppsala, Sweden) using a standard curve generated from the pure insulinconjugate solution. Endogenous rat insulin does not cross-react on thisassay; therefore, any results obtained were due solely to theexogenously administered insulin conjugate and not endogenous ratinsulin.

FIG. 3 shows ˜4× increase in serum insulin concentration from baselinefollowing the intraperitoneal α-methyl-mannose tolerance test(IP(α-MM)TT) indicating α-methyl-mannose-responsive delivery in vivo.Furthermore, very little conjugate was released at physiologicallynormal blood glucose levels during the first two hours of the experimentand virtually no hypoglycemia was induced prior to the introduction ofα-methyl-mannose. However, once the 4× increase in seruminsulin-conjugate concentration induced by the exogenously deliveredα-methyl-mannose, exerted a significant glucose lowering effect.

Example 3 Synthesis of TSB-C4 Framework

A solution of 1,3,5-benzenetricarbonyl chloride (1 gm, 3.8 mmole) indichloromethane (DCM) (5 mL) is added drop-wise to a vigorously stirringsolution of an ω-aminoacid (3.1 equivalents) in 1N NaOH (25 mL) in anice bath. The ice bath is removed and stirring is continued for 4 hoursat room temperature. 2N HCl (˜15 mL) is added dropwise to approximatelypH 2 and the resulting slurry is stirred for an additional 2 hours. Theprecipitate is filtered, washed with cold water (2×20 mL) and dried inair under vacuum and then in a 60 C oven overnight. The resulting whitesolid is used without further purification. Yield for each ω-aminoacid(4-aminobutyric acid: yield 1.6 gm, 91%; 6-aminocaproic acid: yield 1.9gm, 92%)

The above material is taken into DMSO (5 mL) containingN-hydroxysuccinimide (3.1 mmole, 3.1 equiv.) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI, 3.6 mmole, 3.6equiv.) is added at room temperature. The resulting solution is stirredfor 24 hours, diluted with water (125 mL) and extracted with ethylacetate (3×50 mL). The combined organic phase is washed with water (2×50mL), brine (1×50 mL) and dried over MgSO₄. The solvent is evaporated andthe semi-solid residue triturated with acetonitrile (10 mL). The solidis filtered and washed with cold solvent, dried in air under vacuum andthen in a 60 C oven overnight. The product is free of urea bi-product.Benzene-1,3,5-tricarboxy-(N-6-aminocaproic-NHS ester)amide (TSB-C6): 304mg, 36%, mp 140-142 C.Benzene-1,3,5-tricarboxy-(N-4-butyric-NHS-ester)amide (TSB-C4): 245 mg,45%, mp 182-184 C.

Example 4 Synthesis of Azidoethylmannose (AzEM)

a. Synthesis of Bromoethylmannose

DOWEX 50W×4 resin (Alfa Aesar, Ward Hill, Mass.) is washed withdeionized water to remove color. A mixture of 225 gm D-mannose (1.25mol; 1 equiv., Alfa Aesar) and 140 gm DOWEX 50W×4 is treated with 2.2 L2-bromoethanol (30.5 mol, 25 equiv.; 124.97 gm/mol; 1.762 gm/mL; BP=150C; Alfa Aesar) and the stirred mixture heated to 80 C for 4 hours. Thereaction is monitored by TLC (20% methanol/dichloromethane (DCM)).Reaction is complete after about four hours, and then allowed to cool toroom temperature. The solution is filtered to remove the resin, and theresin washed with ethyl acetate and DCM. The resulting filtrate isstripped to an amber oil in a rotory evaporator.

The amber oil is purified on silica gel (4 kg silica packed in DCM) inthe following manner. The crude is dissolved in DCM and loaded onto thecolumn, and then eluted with 2×4 L 10% methanol/DCM; 2×4 L 15%methanol/DCM; and 3×4 L 20% methanol/DCM. Product containing fractions(on the basis of TLC) are pooled and stripped to dryness to afford 152gm of 1-α-bromoethyl-mannose (42%).

b. Conversion of Bromoethylmannose to Azidoethylmannose (AzEM)

A 5 L round bottom three-necked flask, equipped with a heating mantle,an overhead stirrer, and a thermometer, is charged with 150 gmbromoethylmannose (525 mmol). The oil is dissolved in 2 L water andtreated with 68.3 gm sodium azide (1.05 mol, 2 equiv.; 65 gm/mol;Alfa-Aesar) followed by 7.9 gm sodium iodide (52.5 mmol, 0.08 equiv.;149.89 gm/mol; Alfa-Aesar) and the solution warmed to 50 C and stirredovernight. The solution is cooled to room temperature and concentratedto dryness on the rotovap. The solid residue is digested with 3×500 mLof 5:1 vol. CHCl₃:MeOH at 40 C. The combined organic portions arefiltered and evaporated to dryness to afford azidoethylmannose as anoff-white solid.

c. Repurification of Azidoethylmannose

32 gm of azidoethylmannose is taken into 100 mL water. The turbidsolution is filtered through a glass microfibre filter (Whatman GF/B).The filtrate is evaporated to a solid on a rotovapor. The solid is takeninto Methanol (100 mL) and the turbid solution is again filtered througha glass microfibre filter. The resulting pale yellow filtrate isstripped to a solid under vacuum.

The solid is taken into a minimum of methanol (50 mL) and ethyl acetate(150 mL) is added slowly with stirring. The heavy slurry is cooled andfiltered. The solid is air dried (hygroscopic) and put in a 60 C ovenovernight. The Mother Liquor is evaporated under vacuum to a yellow gum.

Example 5 Synthesis of Azidoethylmannobiose (AzEBM)

The AzEM compound from Example 4 is selectively protected using bezenedimethyl ether, purified by column chromatography and subsequentlyreacted with benzyl bromide to give 1-α-(2-azidoethyl)-4,6-benzaldehydediacetal-3-benzyl-mannopyranoside. The product is subsequentlyglycosylated with 1-α-bromo-2,3,4,6-tetrabenzoylmannopyranoside usingsilver triflate chemistry under rigorously anhydrous conditions to givethe protected-azidoethylmannobiose product. The intermediate product isthen deprotected to remove the benzoyl groups to give AzEBM.

Example 6 Synthesis of Aminoethylmannobiose (AEBM)

The azido-terminated compound from Example 5 is readily hydrogenated atroom temperature by using palladium/carbon catalyst, a small amount ofacetic acid, and ethanol as a solvent to give the correspondingamine-terminated compounds. The process is identical to the onedescribed for AETM below, except that those skilled in the art willunderstand that the amounts of reagents, solvents, etc. should be scaledto the number of moles of sugar-ligand to be hydrogenated.

a. Man (α-1,3)-Man(α-1.6)-α-1-aminoethylmannopyranoside(“aminoethyltrimannose”, AETM)

To a solution of 5.3 gm (9.25 mmole)man(α-1,3)-man(α-1.6)-α-1-azidoethylmannopyranoside in 100 mL water and50 mL ethanol was added 0.8 gm 5% Pd/C. The vigorously stirringsuspension was hydrogenated at 30-40 psi for 48 hours or until nostarting material was apparent by TLC (SG, Methanol, SM R_(f) 0.75, PdtR_(f) 0.0, PMA vis.). The suspension was filtered over celite, which wasrinsed with ethanol (2×50 mL) and the filtrate concentrated undervacuum.

HPLC of this material (C18, 3% Acetonitrile/97% 0.1% H₃P0₄, 220 nm, 2ml/min) gave uv adsorption of the injection column void material, Rt 2.5minutes, indicative of benzoate ester.

The filtrate was diluted with 70 mL water and 12 mL of 1N NaOH and thesolution stirred overnight at room temperature (HPLC: no uv material atcolumn void Rt 2.5 min., uv material at Rt 10.5 minutes co-eluting withbenzoic acid). 2 gm of decolorizing charcoal were added and the stirringsuspension heated to 80 C, cooled to room temperature and filtered overcelite. The filtrate pH was adjusted to 8.0 with 2N HCl and thecolorless solution concentrated under vacuum to about 50% volume.

The solution was loaded onto a resin column (Dowex 50 W, 50 gm) andwashed with water until eluting fractions were neutral to pH (6×75 mL)removing any residual acid byproducts. The amine product was washed offthe column with 0.25N ammonium hydroxide (6×75 mL) and the fractionscontaining the amine product-ninhydrin detection were combined andconcentrated to 25-30 mL under vacuum. This concentrated solution wasadded drop-wise to 300 mL stirring ethanol and stirring continued for anadditional 2 hours. The product was filtered, washed with fresh ethanol(2×50 mL) and air dried to a constant weight. The resulting whiteamorphous solid was dried further in a vacuum oven at 80 C for 5 hoursto give 4.1 gm of a white granular solid (TY 5.1 gm). The NMR was cleanof any aromatic protons. ¹H NMR 300 MHz (D₂O) δ 5.08 (s, 1H), 4.87 (s,1H), 4.81 (s, 1H), 4.8-3.6(m, 18H), 2.9(m, 2H).

Example 7 Synthesis of NH₂—B1-BOC2(A1,B29)-Insulin

In a typical synthesis, 4 g of powdered insulin (Sigma Aldrich, St.Louis, Mo.) is dissolved in 100 ml of anhydrous DMSO at room temperaturefollowed by the addition of 4 ml of triethylamine (TEA). The solution isstirred for 30 minutes at room temperature. Next, 1.79 ml (2.6equivalents) of di-tert-butyl-dicarbonate/THF solution (Sigma Aldrich,St. Louis, Mo.) is slowly added to the insulin-TEA solution and mixedfor approximately one hour. The reaction is quenched via the addition of4 ml of a stock solution containing 250 ul of ethanolamine in 5 ml ofDMSO followed by mixing for five minutes. After quenching, the entiresolution is poured into 1600 ml of acetone and mixed briefly with aspatula. Next, 8×400 μl aliquots of a 18.9% HCl:water solution are addeddropwise over the surface of the mixture to precipitate the reactedinsulin. The precipitated material is then centrifuged and thesupernatant decanted into a second beaker while the precipitate cake isset aside. To the supernatant solution, another 8×400 μl aliquots of a18.9% HCl:water solution are added dropwise over the surface of themixture to obtain a second precipitate of reacted insulin. This secondprecipitate is centrifuged and the supernatant is discarded. Thecombined centrifuge cakes from the two precipitation steps are washedonce with acetone followed by drying under vacuum at room temperature toyield the crude powder which typically contains 60% of the desired BOC2product and 40% of the BOC3 material.

A preparative reverse phase HPLC method is used to isolate the pureBOC2-insulin from the crude powder. Buffer A is deionized watercontaining 0.1% TFA and Buffer B is acetonitrile containing 0.1% TFA.The crude powder is dissolved at 25 mg/ml in a 70% A/30% B mixture andsyringe filtered prior to injection on the column. Before purification,the column (Waters SymmetryPrep C18, 7 um, 19×150 mm) is equilibrated at15 ml/minutes with a 70% A/30% B mobile phase using a Waters DeltraPrep600 system. Approximately 5 ml of the crude powder solution is injectedonto the column at a flow rate of 15 ml/minutes over the course of 5minutes after which a linear gradient is employed from 70% A/30% B to62% A/38% B over the course of the next 3.5 minutes and held there foran additional 2.5 minutes. Using this method, the desired BOC2 peakelutes at approximately 10.6 minutes followed closely by the BOC3 peak.Once collected, the solution is rotovapped to remove acetonitrile andlyophilized to obtain pure BOC2-insulin powder. Identity is verified byLC-MS (HT Laboratories, San Diego, Calif.) and site of conjugationdetermined by N-terminal sequencing (Western Analytical, St. Louis,Mo.).

Example 8 Synthesis of Conjugate

The TSB-C4 framework is dissolved at 60 mM in 1.0 ml of anhydrous DMSOfollowed by the addition of 400 ul (excess) of triethylamine (TEA). Thesolution is stirred rapidly for 10 minutes at room temperature. TheNH₂—B1-BOC2(A1,B29)-insulin (MW=6,008 g/mol) is then dissolvedseparately in 7.9 ml of DMSO at a concentration of 7.4 mM. Oncedissolved, the entire drug solution is added dropwise over the course of10 minutes to the framework/DMSO/TEA solution followed by roomtemperature mixing for two hours. The remaining activated esters arethen reacted with the amine-functionalized AEBM affinity ligands in thefollowing manner. A 370 mM solution of affinity ligand is prepared in anappropriate volume of dry DMSO. Once dissolved, enough solution is addedto provide a number of reactive equivalents equal to three times thenumber of initial activated ester groups, N, minus one. For example, ifthere are N=3 initial activated ester groups per framework, then(3×(3−1)×60 mM/370 mM)=0.973 ml of affinity ligand solution are added.If there are N=4 initial activated ester groups per framework, then(3×(4−1)×60 mM/370 mM)=1.46 ml of affinity ligand solution are added,and so on. After the affinity ligand solution is added, the solution isstirred for one more hour at room temperature to ensure completereaction.

The resulting solution is then superdiluted by 10× into a 20 mM pH 5.0HEPES buffered saline solution containing 0.150 M NaCl followed by pHadjustment with dilute HCl to a final pH of 8.0. The aqueous solution isfirst purified by size exclusion using an appropriate solid phase forthe desired separation of conjugated and unconjugated materials. Thesolution passing through the column void volume is then concentratedusing an appropriately sized ultrafiltration membrane to approximately10 ml. This solution is further purified to obtain the desired productusing preparative reverse phase HPLC on a Waters C8, 7 um, 19×150 mmcolumn. Buffer A is deionized water containing 0.1% TFA and Buffer B isacetonitrile containing 0.1% TFA. Before purification, the column isequilibrated at 15 ml/minutes with a 80% A/20% B mobile phase using aWaters DeltraPrep 600 system. Approximately 5 ml of the crude solutionis injected onto the column over the course of 2 minutes at a flow rateof 15 ml/minutes after which a linear gradient is employed from 80%A/20% B to 75% A/25% B over the next 5 minutes followed by a slowerlinear gradient from 75% A/25% B to 62% A/38% B over the next 22minutes. The retention time of the desired peak will vary depending onthe drug, framework, and affinity ligand used. Once collected, thesolution is rotovapped to remove acetonitrile and lyophilized to obtainpure conjugate whose identity may be verified by LC-MS (HT Laboratories,San Diego, Calif.). Because the starting NH₂—B1-BOC2(A1,B29)-insulinmaterial only possesses one free amine group at the Phe-B1 terminus, thePhe-B1 is the only site of insulin conjugation to the framework asverified in each deprotected final product by N-terminal sequencing.

Example 9 Conjugates of Formula (I)

This example describes some exemplary conjugates of formula (I):

Yet other embodiments of these conjugates as well as intermediates andmethods of making these conjugates can be found in U.S. ProvisionalApplication No. 61/162,105 filed Mar. 20, 2009 and corresponding PCTapplication filed on Jan. 27, 2010. The entire contents of these relatedapplications are incorporated herein by reference.

In certain embodiments, a conjugate of formula (I) may include one ormore of the following exemplary groups:

R^(x)

In certain embodiments, R^(x) is hydrogen. In certain embodiments, R^(x)is optionally substituted C₁₋₆ alkyl. In certain embodiments, R^(x) isoptionally substituted C₁₋₃ alkyl. In certain embodiments, R^(x) isoptionally substituted methyl. In certain embodiments, R^(x) is —CH₃.

Z¹

In certain embodiments, Z¹ is an optionally substituted bivalent C₁₋₁₀,C₁₋₈, C₁₋₆, C₁₋₄, or C₁₋₂ hydrocarbon chain. In certain embodiments, Z¹is —(CH₂)—, —(CH₂CH₂)—, —(CH₂CH₂CH₂)—, —(CH₂CH₂CH₂CH₂)—,—(CH₂CH₂CH₂CH₂CH₂)—, or —(CH₂CH₂CH₂CH₂CH₂CH₂)—. In certain embodiments,Z¹ is —(CH₂)— or —(CH₂CH₂)—. In certain embodiments, Z¹ is —(CH₂)—. Incertain embodiments, Z¹ is —(CH₂CH₂)—. In certain embodiments, Z¹ is—(CH₂CH₂CH₂)—. In certain embodiments, Z¹ is —(CH₂CH₂CH₂CH₂)—.

In certain embodiments, Z¹ is an optionally substituted bivalent C₁₋₁₀hydrocarbon chain, wherein 1, 2 or 3 methylene units of Z¹ areoptionally and independently replaced with one or more groups selectedfrom —S—, —O—, —NR^(a)—, —(C═NR^(a))—, —(C═O)—, —(S═O)—, —S(═O)₂—,—(CR^(b)═CR^(b))—, —(N═N)—, an optionally substituted arylene moiety oran optionally substituted heteroarylene moiety. In certain embodiments,Z¹ is an optionally substituted bivalent C₁₋₁₀ hydrocarbon chain,wherein 1, 2 or 3 methylene units of Z¹ are optionally and independentlyreplaced with one or more groups selected from —S—, —O—, —NR^(a)—,—(C═NR^(a))—, or —(C═O)—. In certain embodiments, Z¹ is—CH₂CH₂NH(C═O)C(CH₃)₂—, —CH₂CH₂N(C═NH)(CH₂)₃S—, —CH(R^(f))₂,—CH₂CH(R^(f))₂, —CH₂CH₂CH(R)₂—, —CH₂S—, or —CH₂CH₂S—, wherein R^(f) isoptionally substituted aliphatic, optionally substitutedheteroaliphatic, optionally substituted aryl, optionally substitutedheteroaryl (e.g., in certain embodiments, R^(f) is optionallysubstituted aryl; in certain embodiments, R^(f) is phenyl). In certainembodiments, Z¹ is —CH₂CH₂NH(C═O)C(CH₃)₂— or —CH₂CH₂N(C═NH)(CH₂)₃S—. Incertain embodiments, Z¹ is —CH₂CH₂NH(C═O)C(CH₃)₂—. In certainembodiments, Z¹ is —CH₂CH₂N(C═NH)(CH₂)₃S—.

Y¹

In certain embodiments, Y¹ is a fragment of a free radical initiator.Such a fragment is encompassed by the definition of Y¹, as initiatorfragments may include halogen, —OR^(e), —SR^(e), optionally substitutedaliphatic, optionally substituted heteroaliphatic, optionallysubstituted aryl, and optionally substituted heteroaryl moieties.

In certain embodiments, Y¹ is hydrogen, halogen, or an initiatorfragment. In certain embodiments, Y¹ is hydrogen or halogen. In certainembodiments, Y¹ is hydrogen or bromine.

X¹

In certain embodiments, X¹ is —OR^(c). In certain embodiments, X¹ is amixture of —OR^(c) and —N(R^(d))₂. In certain embodiments, X¹ is—N(R^(d))₂.

W¹ and

In certain embodiments,

is a single covalent bond.

In certain embodiments, W¹ is covalently bound to the polymer via anamino group. In certain embodiments, W¹ is covalently bound to thepolymer via a primary amino group.

For example, in certain embodiments, the group

corresponds to the group

wherein the group [Drug-NH—] or [Drug-N═] is the drug directlycovalently conjugated via a primary amino group. In other embodiments,the drug may include a spacer group (e.g., an alkylene group, arylenegroup, heteroarylene group, ester linkage, amide linkage, and the like)which terminates with a pendant amino group. The latter embodimentsenable greater separation between the active portion of the drug and thepolymer.r

In certain embodiments, r is an integer between 10-25, inclusive. Incertain embodiments, r is an integer between 15-25, inclusive. Incertain embodiments, r is an integer between 20-25, inclusive. Incertain embodiments, r is an integer between 5-20, inclusive. In certainembodiments, r is an integer between 10-20, inclusive. In certainembodiments, r is an integer between 15-20, inclusive. In certainembodiments, r is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24 or 25. In certain embodiments r is 5. In certainembodiments r is 10. In certain embodiments r is 15. In certainembodiments r is 20. In certain embodiments r is 25.

In certain embodiments, the group:

corresponds to a mixture of the groups:

wherein the sum of (g+t) is equal to r. In certain embodiments, eachinstance of g and t is, independently, an integer between 1 and 24,inclusive, with the proviso that the sum of (g+t) is greater than orequal to 5 and less than or equal to 25. In certain embodiments, g and tare present in a ratio of about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3,1:2, or 1:1 (g to t). In certain embodiments, t and g are present in aratio of about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, or 1:2 (t to g).

Exemplary Conjugates

In certain embodiments, a conjugate of formula (I-a1) may be used:

In certain embodiments, a conjugate of formula (I-a2) may be used:

In certain embodiments, a conjugate of formula (I-b1) may be used:

In certain embodiments, a conjugate of formula (I-b2) may be used:

In certain embodiments, a conjugate of formula (I-c1) may be used:

In certain embodiments, a conjugate of formula (I-c2) may be used:

In any of these exemplary conjugates, the group:

may correspond to a mixture of the groups:

wherein the sum of (g+t) is equal to r, respectively. In certainembodiments, r is 10. In certain embodiments, r is 20.

Characterization of Conjugates

The conjugates can be characterized by any analytical method includingnuclear magnetic resonance (e.g., ¹H NMR); gel permeation chromatography(GPC) for molecular weight and polydispersity; and Fourier transforminfrared spectroscopy (FTIR) or acid titration for determination of thenumber of acid groups per chain.

In certain embodiments the conjugate framework (i.e., without includingthe affinity ligands or drug) has a molecular weight of less than 10,000Da, e.g., in the range of about 100 to about 10,000 Da. In certainembodiments, the conjugate framework has a molecular weight in the rangeof about 300 to about 5,000 Da. In certain embodiments, the conjugateframework has a molecular weight in the range of about 500 to about2,500 Da. In certain embodiments, the conjugate framework has amolecular weight in the range of about 1,000 to 2,000 Da. In certainembodiments, the conjugate framework has a molecular weight in the rangeof about 200 to 1,000 Da. In certain embodiments, the conjugateframework has a molecular weight in the range of about 300 to 800 Da.

In certain embodiments, a mixture of conjugates is generated. Theconjugates in this mixture may have the same or different molecularweights. In one embodiment, the polydispersity of the mixture is lessthan 1.5. In one embodiment, the polydispersity of the mixture is lessthan 1.25.

Example 10 Conjugates of Formula (II)

This example describes some exemplary conjugates of formula (II):

Yet other embodiments of these conjugates as well as intermediates andmethods of making these conjugates can be found in U.S. ProvisionalApplication No. 61/147,878 filed Jan. 28, 2009, U.S. ProvisionalApplication No. 61/159,643 filed Mar. 12, 2009, U.S. ProvisionalApplication No. 61/162,107 filed Mar. 20, 2009, U.S. ProvisionalApplication No. 61/163,084 filed Mar. 25, 2009, U.S. ProvisionalApplication No. 61/219,897 filed Jun. 24, 2009, U.S. ProvisionalApplication No. 61/223,572 filed Jul. 7, 2009, U.S. ProvisionalApplication No. 61/252,857 filed Oct. 19, 2009, and corresponding PCTapplication filed on Jan. 27, 2010. The entire contents of these relatedapplications are incorporated herein by reference.

In some embodiments, the present disclosure provides conjugates ofgeneral formula (II-a1):

For example, in some embodiments, the present disclosure providesconjugates of formula:

In some embodiments, the present disclosure provides conjugates ofgeneral formula (II-a2):

For example, in some embodiments, the present disclosure providesconjugates of formula:

In some embodiments, the present disclosure provides conjugates ofgeneral formula (II-a3):

For example, in some embodiments, the present disclosure providesconjugates of formula:

Characterization of Conjugates

The conjugates can be characterized by any analytical method includingnuclear magnetic resonance (e.g., ¹H NMR); gel permeation chromatography(GPC) for molecular weight and polydispersity; Fourier transforminfrared spectroscopy (FTIR), etc.

In certain embodiments the conjugate framework (i.e., without includingthe affinity ligands or drug) has a molecular weight of less than 10,000Da, e.g., in the range of about 100 to about 10,000 Da. In certainembodiments, the conjugate framework has a molecular weight in the rangeof about 300 to about 5,000 Da. In certain embodiments, the conjugateframework has a molecular weight in the range of about 500 to about2,500 Da. In certain embodiments, the conjugate framework has amolecular weight in the range of about 1,000 to 2,000 Da. In certainembodiments, the conjugate framework has a molecular weight in the rangeof about 200 to 1,000 Da. In certain embodiments, the conjugateframework has a molecular weight in the range of about 300 to 800 Da.

Example 11 Synthesis of Azidophenyl-Sugar Modified Con A

This example and those that follow describes the preparation of someexemplary binding-site modified lectins that could be used to prepare amaterial of the present disclosure.

All steps were performed at room temperature unless otherwise specified.First, 5.0 g of native Con A (Sigma-Aldrich, St. Louis, Mo.) wasdissolved in 200 ml of a 10 mM pH 5.0 acetate buffer solution containing150 mM sodium chloride, 2 mM calcium chloride, 2 mM manganese chloride,and 0.1% w/v sodium azide (S28 buffer) and any insoluble material wasseparated by centrifugation and/or filtration. We have found thatdifferent commercial preparations of native Con A contain appreciableconcentrations of inhibitory sugars that are, in certain embodiments,removed prior to photoaffinity modification. To that end, the solutionwas purified through a Biogel-P6 size exclusion column with an S28mobile phase two times. Finally, the resulting solution was diluted withS28 to a final volume of 1 L. Under gentle stirring conditions, 0.4 g ofhydroquinone (Sigma-Aldrich, St. Louis, Mo.) was added followed by 165mg of either azidophenylglucose (APG, PolyOrg Inc., Leominster, Mass.)or azidophenylmannose (APM, PolyOrg Inc., Leominster, Mass.). Thesolution was stirred in the dark at 4 C for one hour at the lowestpossible stir speed. After one hour of stirring, any additionalinsoluble material was removed via centrifugation and/or filtration. 200ml of the solution was poured into a 9″×13″ aluminum pan and reacted at4 C inside a CL-1000 UV crosslinking oven (UVP, Upland, Calif.) for 15min at 360 nm (the UV reaction may also take place using 302 nm light).Following the reaction, any additional insoluble material was removedvia centrifugation and/or filtration. The clarified solution was thenpurified 1× through Biogel-P6 size exclusion columns (Econopak, Bio-RadLabs, Hercules, Calif.) with an S28 mobile phase. The UV crosslinkingreaction and P6 purification process was then repeated until the entiresolution was reacted. Finally, the combined P6-purified solutions wereconcentrated down to ˜180 ml using a Pall tangential flow filtrationcartridge apparatus (Millipore, Billerica, Mass.) equipped with Omega30K membranes. The resulting solution was clarified via centrifugationand/or filtration and passed through 0.22 um filters prior to affinitycolumn purification.

Example 12 Generalized Synthesis of Diazirine Photoreactive Ligands

0.9 mmol of aminoethyl (AE) functionalized sugar ligand (e.g., AEG, AEM,AEBM, AETM) were dissolved in 4 ml of anhydrous DMSO after which 1.6 mlof anhydrous triethylamine (TEA) were added to form a cloudy emulsion.In a separate container, 200 mg (0.9 mmol) of NHS-diazirine (ThermoFisher Scientific Inc., Rockford, Ill.) powder was dissolved in 4 ml ofanhydrous DMSO under dark conditions. Once dissolved, the NHS-diazirinesolution was added dropwise to the AE-sugar solution and then allowed toreact overnight at room temperature in the dark. TLC analysis (50%ethanol:50% ethyl acetate) of the overnight solution confirmed completereaction as evidenced by the co-elution of the UV signal of thediazirine moiety (254 nm) and the sugar signal (sulfuric acid-ethanolstain) and concomitant disappearance of the AE-functionalized sugarligand from the origin of the TLC (sulfuric acid-ethanol stain). Thesolution was then diluted into 80 ml of a pH 5.0, 25 mM HEPES solutioncontaining 0.15 M sodium chloride, pH adjusted to pH 5 if necessary, andthen frozen until required for photoaffinity reaction with Con A.

Example 13 Synthesis and Characterization of Sugar-FunctionalizedDiazirine Con A

All steps were performed at room temperature unless otherwise specified.First, 5.0 g of native Con A (Sigma-Aldrich, St. Louis, Mo.) wasdissolved in 200 ml of a 10 mM pH 5.0 acetate buffer solution containing150 mM sodium chloride, 2 mM calcium chloride, 2 mM manganese chloride,and 0.1% w/v sodium azide (S28 buffer) and any insoluble material wereseparated by centrifugation and/or filtration. We have found thatdifferent commercial preparations of native Con A contain appreciableconcentrations of inhibitory sugars that are, in certain embodiments,removed prior to photoaffinity modification. To that end, the solutionwas purified through a Biogel-P6 size exclusion column with an S28mobile phase two times. Finally, the resulting solution was diluted withS28 to a final volume of 1 L. Next, the solution volume was brought upto 1 L—⅓ ligand volume, using 1×S28 and poured into a 1 L media bottlewith stir bar. Under gentle stirring conditions in the dark, 0.4 g ofhydroquinone (Sigma-Aldrich, St. Louis, Mo.) was dissolved. Next, 33 mlof the diazirine-sugar conjugate obtained in Example 43 was added in 7aliquots under gentle stirring conditions in the dark. Once dissolved,the entire solution was incubated under gentle stirring for anadditional 10 min at 4 C in the dark. After 10 min of stirring, anyadditional insoluble material was removed via centrifugation and/orfiltration. 250 ml of the solution was poured into a 9″×13″ aluminum panand reacted at 4 C inside a CL-1000 UV crosslinking oven (UVP, Upland,Calif.) for 15 min at 360 nm. Following the reaction, any additionalinsoluble material was removed via centrifugation and/or filtration. Theclarified solution was then purified 1× through Biogel-P6 size exclusioncolumns (Econopak, Bio-Rad Labs, Hercules, Calif.) with an S28 mobilephase. The UV crosslinking reaction and P6 purification process was thenrepeated until the entire solution was reacted. Finally, the combinedP6-purified solutions were concentrated down to ˜180 ml using a Palltangential flow filtration cartridge apparatus (Millipore, Billerica,Mass.) equipped with Omega 30K membranes. The resulting solution wasclarified via centrifugation and/or filtration and passed through 0.22um filters prior to affinity column purification.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

1. A cross-linked material comprising: multivalent cross-linking agentsthat bind an exogenous target molecule; and conjugates that include adrug and two or more separate affinity ligands bound to a conjugateframework, wherein the two or more affinity ligands compete with theexogenous target molecule for binding with the cross-linking agents andwherein conjugates are cross-linked within the material as a result ofnon-covalent interactions between cross-linking agents and affinityligands on different conjugates.
 2. The material of claim 1, wherein theexogenous target molecule includes a saccharide.
 3. The material ofclaim 2, wherein the exogenous target molecule is α-methyl-mannose. 4.(canceled)
 5. The material of claim 1, wherein the affinity ligands ofthe conjugates include a saccharide selected from glucose, mannose,glucosamine, mannosamine, methylglucose, methylmannose, ethylglucose,and ethylmannose.
 6. The material of claim 1, wherein the affinityligands of the conjugates include a bimmanose or trimannose.
 7. Thematerial of claim 1, wherein the affinity ligands of the conjugatesinclude aminoethylglucose (AEG), aminoethylmannose (AEM),aminoethylbimannose (AEBM) or aminoethyltrimannose (AETM). 8-10.(canceled)
 11. The material of claim 1, wherein the multivalentcross-linking agents include a lectin. 12-14. (canceled)
 15. Thematerial of claim 11, wherein the lectins are covalently bonded to arecognition element, wherein the recognition element competes with theexogenous target molecule and affinity ligands of the conjugate forbinding to the lectin, and the lectin has a higher affinity for theaffinity ligands of the conjugate than for the recognition element. 16.The material of claim 15, wherein the exogenous target molecule is asaccharide and both the affinity ligands of the conjugate and therecognition element include a saccharide. 17-18. (canceled)
 19. Thematerial of claim 1, wherein the multivalent cross-linking agentsinclude a peptide aptamer.
 20. The material of claim 1, wherein themultivalent cross-linking agents include a polynucleotide aptamer. 21.(canceled)
 22. The material of claim 1, wherein the drug is an insulinmolecule. 23-29. (canceled)
 30. The material of claim 1, wherein theconjugate is of the general formula:

wherein: each occurrence of

represents a potential branch within the conjugate; each occurrence of

represents a potential repeat within a branch of the conjugate; eachoccurrence of

is independently a covalent bond, a carbon atom, a heteroatom, or anoptionally substituted group selected from the group consisting of acyl,aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; eachoccurrence of T is independently a covalent bond or a bivalent, straightor branched, saturated or unsaturated, optionally substituted C₁₋₃₀hydrocarbon chain wherein one or more methylene units of T areoptionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—,—C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)₂—, —N(R)SO₂—,—SO₂N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group;each occurrence of R is independently hydrogen, a suitable protectinggroup, or an acyl moiety, arylalkyl moiety, aliphatic moiety, arylmoiety, heteroaryl moiety, or heteroaliphatic moiety; —B is -T-L^(B)-X;each occurrence of X is independently an affinity ligand; eachoccurrence of L^(B) is independently a covalent bond or a group derivedfrom the covalent conjugation of a T with an X; -D is -T-L^(D)-W; eachoccurrence of W is independently a drug; each occurrence of L^(D) isindependently a covalent bond or a group derived from the covalentconjugation of a T with a W; k is an integer from 2 to 11, inclusive,defining at least two k-branches within the conjugate; q is an integerfrom 1 to 4, inclusive; k+q is an integer from 3 to 12, inclusive; eachoccurrence of p is independently an integer from 1 to 5, inclusive; andeach occurrence of n is independently an integer from 0 to 5, inclusive;and each occurrence of m is independently an integer from 1 to 5,inclusive; and each occurrence of v is independently an integer from 0to 5, inclusive, with the proviso that within each k-branch at least oneoccurrence of n is ≧1 and at least one occurrence of v is ≧1. 31-40.(canceled)
 41. A method comprising administering a material of claim 1to a patient and subsequently administering a triggering amount of theexogenous target molecule to the patient. 42-61. (canceled)